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-mediated bovine pulmonary endothelial cell apoptosis
Division of Pulmonary and Critical Care Medicine, Department of Medicine, and
# Departments of Anesthesiology and Critical Care Medicine, Cell Biology, and Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; and
* Department of Medicine, Albert Einstein College of Medicine, New York, New York, USA
1Correspondence: Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Room 4B.77, Baltimore, MD 21224-6801, USA. E-mail: drgarcia{at}jhmi.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-induced endothelial cell apoptosis. As these events reflect the activation of the multifunctional endothelial cell (EC) MLCK isoform, we assessed the direct role of EC MLCK in the regulation of TNF--induced apoptosis. Bovine pulmonary artery endothelial cells expressing either an adenovirus encoding antisense MLCK cDNA (Ad.GFP-AS MLCK) or a dominant/negative EC MLCK construct (EC MLCK-ATPdel) resulted in marked reductions in MLCK activity and TNF--mediated apoptosis. In contrast, a constitutively active EC MLCK lacking the carboxyl-terminal autoinhibitory domains (EC MLCK-1745) markedly enhanced the apoptotic response to TNF-. Immunostaining in GFP-EC MLCK-expressing cells revealed colocalization of caspase 8 and EC MLCK along actin stress fibers after TNF-. TNF- induced the caspase-dependent cleavage of EC MLCK-1745 in transfected endothelial cells, which was confirmed by mass spectroscopy with in vitro cleavage by caspase 3 at LKKD (D1703). The resulting MLCK fragments displayed significant calmodulin-independent kinase activity. These studies convincingly demonstrate that novel interactions between the apoptotic machinery and EC MLCK exist that regulate the endothelial contractile apparatus in TNF--induced apoptosis.—Petrache, I., Birukov, K., Zaiman, A. L., Crow, M. T., Deng, H., Wadgaonkar, R., Romer, L. H., Garcia, J. G. N. Caspase-dependent cleavage of myosin light chain kinase (MLCK) is involved in TNF--mediated bovine pulmonary endothelial cell apoptosis.
Key Words: cytoskeleton • stress fibers • Rho kinase • blebbing • autoinhibitory domain
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PRODUCTION and augmented cellular apoptosis are characteristic features of diverse inflammatory processes, including acute lung injury (1
, 2)
. TNF- directly induces pulmonary endothelial cell apoptosis in vivo and in vitro through the cytoplasmic death domain (DD) of the TNF- receptor 1 (TNFR1) and its association with adaptor proteins such as the TNF receptor-associated death domain protein (TRADD), receptor interacting protein-1 (RIP1), and TNF receptor-associated factor 2 (TRAF2). TRADD in turn recruits Fas-associated death domain protein (FADD) to the death-inducing signaling complex (DISC), leading to the sequential recruitment and activation of the initiator caspase 8 (3
, 4)
. Caspase 8 activates the executioner caspase cascade (caspases 3, 6, and 7), which cleave key cellular substrates, thus annihilating important survival pathways and creating the typical morphologic changes of membrane blebbing, cellular shrinkage, and apoptotic body formation. A growing number of cytoskeletal components are recognized as caspase substrates and alterations in actin assembly/disassembly are required for the initiation of membrane blebbing, a recognized feature of apoptotic cell death. There is increasing appreciation, however, that the actin cytoskeleton may participate not only in the terminal stages of apoptotic cell death, but also in the signaling pathways involved in the decision to undergo apoptosis. Using pharmacologic inhibitors, we recently demonstrated that TNF--induced changes in the endothelial cytoskeleton mediated by myosin light chain (MLC) phosphorylation are critical for the morphological changes that occur downstream of caspase activation. The Ca2+/calmodulin-dependent endothelial cell MLC kinase (MLCK) isoform catalyzes the MLC phosphorylation with coordinate inactivation of myosin phosphatase via the Rho GTPase effector Rho kinase. Both MLCK and Rho kinase have been implicated in the development of plasma membrane blebbing in several cell lines (5
, 6)
, and Rho kinase was recently identified as a substrate for caspase 3 with cleavage generating a constitutively active fragment of the enzyme (6)
. Although the smooth muscle (SM) MLCK isoform differs considerably from the larger EC MLCK isoform (7)
, a recent report in an immortalized epithelial cell line overexpressing SM MLCK suggested that MLCK may regulate apoptosis by facilitating TNFR1 recruitment to the plasma membrane (8)
. Indeed, our preliminary observations in a TNF- model of endothelial apoptosis suggested potential EC MLCK involvement in events that precede caspase activation.
In this study, using complementary molecular approaches with targeted MLCK mutants and in vitro mass spectroscopy of cleavage products, we demonstrate that EC MLCK is a critical participant in the regulation of TNF--induced actin microfilament changes and endothelial cell apoptosis. Our results indicate that the regulatory effects of MLCK occur early in the apoptotic pathway in conjunction with novel initiator caspase 8 activation and colocalization with EC MLCK along actin stress fibers. Our data strongly confirm direct caspase-EC MLCK involvement with caspase cleavage resulting in the generation of a constitutively active MLCK fragment. These results confirm the critical participation of the cytoskeleton in the regulation of endothelial cell apoptosis possibly via control of cellular localization of specific components of the apoptotic machinery.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(biological activity of 2 x 107 U/mg) and anti-MLCK antibody (K36) were purchased from Sigma Aldrich (St. Louis, MO, USA). Texas red-X phalloidin and secondary antibodies conjugated to immunofluorescent dyes were purchased from Molecular Probes, Inc. (Eugene, OR, USA). These studies used antibodies raised in rabbits against the di-phosphorylated form of MLC (Ser19, Thr18) described previously in detail (9)
. Human recombinant caspase 3 and DAPI (4’,6-diamidine-2’-phenylindole dihydrochloride) were obtained from Calbiochem-Novalbiochem Corp. (La Jolla, CA, USA). Z-DEVD-fmk, a specific caspase 3 inhibitor was purchased from Alexis Biochemicals (San Diego, CA, USA). Anti-caspase 8 antibody was obtained from Novocastra (Vector Laboratories, Burlingame, CA, USA).
MLCK constructs and deletion mutants
Full-length EC MLCK1 obtained from an endothelial cDNA library (10
11
12)
was used as a template for PCR to obtain the deletion mutants EC MLCK-1745 and EC MLCK-ATPdel. EC MLCK-1745 lacks the carboxyl-terminal amino acids #1745–1914, encoding the autoinhibitory domains of the protein (12)
. Deletion of these same amino acids in the SM MLCK isoform results in a constitutively activated mutant (13)
. EC MLCK-ATPdel lacks amino acids #1580–1607, which reside within the catalytic core of the enzyme and include the ATP binding site, resulting in a kinase-dead mutation. Both constructions were cloned into pcDNA3.1/V5/TOPO (Invitrogen Corporation), thereby introducing a V5 epitope tag at the carboxyl-terminal ends. In specific experiments, we used an engineered p-GFP EC MLCK2 construct that is driven by a CMV promoter and contains the amino-terminal GFP-tagged EC-MLCK splice variant 2, which is identical to the full-length isoform except for the deletion of exon 8 (amino acids #436–506 due to alternative splicing) (12)
. A recombinant adenovirus encoding the 5' 2.8 kb fragment of EC MLCK1 cDNA in reverse orientation (Ad.GFP-AS MLCK) was constructed in the pAdTrack CMV shuttle vector. Recombination and recombinant virus production was performed using the method of He et al. (14)
. The initial 5' 2.8 kb fragment of EC MLCK1 was removed by digesting pFasBAC/MLCK1 with KpnI, blunting the ending site with Klenow, followed by digestion with EagI. This 2.8 kb fragment was isolated and cloned with NotI and EcoRV. After confirmation and purification, the plasmid, was cut with PmeI and transformed into BJ5183 containing pAdeasy1. Recombinant viruses were isolated on kanamycin plates and verified by restriction analysis.
Transfection and infection
Endothelial cells were transiently transfected in 12-well tissue culture plates at 50% confluence. For each transfection, 1 µg plasmid DNA was incubated with 6 µL Fugene (Roche Molecular Biochemicals, Indianapolis, IN, USA) in 50 µL serum-free Optimem (Gibco, Invitrogen) at room temperature for 15 min, followed by addition of 450 µL of serum-free Optimem (Gibco) to each tube containing the lipid–DNA complexes. The mixture was overlaid onto the cells, which were incubated for 4 h at 37°C, followed by addition of complete media (1:3 volumes; 48 h). Adenoviral infection of cells was performed at a multiplicity of infection of 30–40 into 50–70% confluent endothelium in a minimal volume of low-serum media (2%). After 20 h of incubation, medium was completely removed and the cells washed and incubated for 24–48 h before initiating the experiments described below.
Immunofluorescent staining of the endothelial cytoskeleton and caspases
Bovine pulmonary artery endothelial cells were cultured to confluence on coverslips coated with 1% gelatin. After exposure to experimental conditions, endothelial cell monolayers were fixed in 3.7% formaldehyde and permeabilized with 0.25% Triton X-100. After staining, coverslips were mounted on slides and examined under oil immersion using an Eclipse TE300 inverted microscope (Nikon Inc., Melville, NY, USA) connected to a digital camera linked to an image processor. Actin was visualized by Texas red-phalloidin staining (1:200) for 1 h at room temperature, enabling examination of endothelial cell morphology (cellular rounding, shrinkage), intercellular gap formation, and intracellular actin filament reorganization (stress fiber formation, cortical, or perinuclear actin organization). Immunoreactive staining for cleaved caspase 7 or for caspase 8 was performed after fixation and permeabilization at room temperature with 0.5% Triton X-100 in 3% paraformaldehyde (2 min), followed by 3% paraformaldehyde alone (20 min) and washing with PBS. The cells were then incubated with blocking solution (2% BSA in PBS, 1 h) with the primary antibody (1 h), followed by three washes with PBS-Tween (0.1%) and incubation for 1 h with an appropriate secondary antibody conjugated to immunofluorescent dyes (Alexa 488 for green fluorescence and Alexa 546 or Texas red for red fluorescence). After three washes with PBS-Tween (0.1%), the coverslips were mounted and analyzed using Nikon video-imaging system as described above. In some experiments (see Fig. 5A
), the images were obtained with an epifluorescence inverted microscope (Nikon Eclipse TE-200) connected to a digital camera. Images were acquired and processed using Openlab software (Improvision, Lexington, MA, USA) linked to an image processor. All images were recorded and saved in Adobe Photoshop.
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Western immunoblotting
Endothelial cell proteins were separated by SDS-PAGE, transferred to Immobilon PVDF membrane (Millipore, Bedford, MA, USA) and immunoblotted for 1 h with the primary antibody as described previously (9)
, followed by addition of the appropriate horseradish peroxidase-conjugated secondary antibody (1:10,000). The reaction was visualized by enhanced chemiluminescence or chemifluorescence and autoradiography (Amersham, Arlington Heights, IL, USA), according to the manufacturer’s instructions.
Apoptosis assays
Caspase 3 activity was assessed with the Caspase-3 Intracellular Activity Assay Kit II (PhiPhiLux G2D2) from Calbiochem-Novalbiochem by incubating the endothelial monolayer with a specific caspase 3 substrate conjugated to two fluorophores. The activity of caspase 3 could then be visualized with an Eclipse TE300 inverted microscope (Nikon Inc.) as intense red fluorescence at 552 nm and 580 nm wavelengths. Caspase 7 activity was determined by immunofluorescent imaging with a specific anti-cleaved (active) caspase 7 antibody (BD Transduction Laboratories, Lexington, KY). DAPI staining: endothelial cells were cultured to confluence in 12-well dishes on coverslips coated with gelatin. After exposure to experimental conditions, endothelial cell monolayers were fixed in 3.7% formaldehyde, pretreated in a solution containing 2.1 g citric acid and 0.5 mL Tween-20 in 100 mL distilled H2O, and stained with DAPI (0.2 mg DAPI, 11.8 g citric acid 100 mL of distilled H2O). After staining, coverslips were mounted on slides and examined under oil immersion at 461 nm using an Eclipse TE300 inverted microscope for typical apoptotic nuclear changes such as chromatin margination and nuclear condensation.
In vitro caspase 3 cleavage assay
Purified recombinant EC MLCK1 or the SM MLCK isoform obtained using a baculovirus synthesis system (7)
was incubated with active human recombinant caspase 3 at 37°C in the presence or absence of a caspase 3 inhibitor, Z-VAD-fmk or Z-DEVD-fmk. The reaction was terminated by the addition of Laemmli sample buffer. The samples were boiled for 5 min and electrophoresed on SDS gels, followed by either Western blot using anti-MLCK antibody or Coomassie staining.
In vitro MLCK activity assay
Baculovirus-expressed SM MLC was used as a substrate after His-tag excision by rTEV protease as we have described previously (7)
. Purified MLCK-1 and SM MLCK were diluted in 50 mM MOPS, pH-7.4, 10 mM Mg2+-acetate, 0.05% 2-mercaptoethanol containing 1 mg/mL bovine serum albumin to a 1.25 x 10–11 M final assay concentration. MLCK activity was determined by measuring 32P incorporation into the regulatory MLC used as substrate (7)
. The MLCK activity assays were performed in 50 mM MOPS, pH 7.4, 10 mM Mg2+-acetate, 0.025% 2-mercaptoethanol in the presence of 0.3 mM CaCl2, 10-6 M calmodulin, 10-7 M [
-32P]-ATP at 0.5 Ci/mmol specific activity, and 1.25–15 x 10-6 M myosin light chain at 22°C as described previously (7
, 15)
. Results are expressed relative to the activity of the control sample for each experiment, which was MLCK-1 in Ca2+-containing buffer for the MLCK-1 samples and SM MLCK in Ca2+-containing buffer for the SM MLCK samples. A two-tailed Student’s t test was used to determine statistical significance.
Mass spectroscopy analysis
The bands containing MLCK and its fragments after caspase cleavage were excised from SDS-polyacrylamide gels and completely destained with 200 mM ammonium bicarbonate in 50% acetonitrile. These gel fragments were then treated with 10 mM DTT in 0.1 M ammonium bicarbonate for protein reduction. Free cysteine residues were alkylated with freshly prepared 55 mM iodoacetamide in 0.1 M ammonium bicarbonate. Tryptic digestion was started with the addition of 25 ng/µl Sequence Grade Modified Trypsin (Promega) in ammonium bicarbonate buffer. The protein was digested for at least 16 h at 30°C with agitation. The cyanogen bromide (CNBr) cleavage was performed for at least 14 h in the dark at room temperature by adding 25 µL of CNBr in 70% formic acid. The digestion products were cleaned and concentrated using micro-C18 ZipTip (Millipore), mixed with 0.5 µL of 10 mg mL-1 -cyano-4-hydroxysuccinnamic acid in 50% acetonitrile, 0.1% (v/v) TFA, and applied onto a MALDI plate. MALDI mass spectra were recorded with a PerSeptive Voyager-DE STR MALDI time-of-flight mass spectrometer operated in the reflectron mode. In general, the mass measurement accuracy with internal calibration was > 100 ppm.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-induced endothelial apoptosis and caspase activation-induced endothelial cell apoptosis and have provided strong evidence that important cross-talk exists between the apoptotic caspase pathway and the microfilament cytoskeleton (9)
. To clarify the role of EC MLCK in TNF--induced apoptosis, we used two strategies to reduce MLCK activity: infection with an adenovirus encoding MLCK in the antisense orientation (GFP-AS MLCK) and transfection with a catalytically inactive, dominant negative MLCK (EC MLCK-ATPdel) in which the ATP binding site is deleted (kinase-dead). Figure 1
demonstrates that compared with vehicle controls (Fig. 1A
), exposure to TNF- (20 ng/mL, 3–4 h) produces prominent actin microfilament rearrangement with a dramatic increase in stress fiber formation and development of intercellular gaps (Fig. 1C, E
, arrowheads). TNF--induced increases in actin stress fiber formation were markedly attenuated by overexpression of either the antisense MLCK oligonucleotides (GFP-AS MLCK) (Fig. 1C, D
, arrows) or the inactive EC MLCK-ATPdel (data not shown), but not by the GFP empty vector (Fig. 1E, F
). These experiments confirm the requirement for MLCK catalytic activity in TNF--induced endothelial cell microfilament rearrangement. Western blot with an antibody that recognizes only diphosphorylated MLCs demonstrated that the reduction in MLCK gene expression by this strategy dramatically decreases MLC phosphorylation under basal conditions and in endothelial cells activated by TNF- (20 ng/mL, 1 h), a process known to trigger MLCK-dependent MLC phosphorylation (9)
(Fig. 2
A). Apoptosis in endothelial cells infected with GFP-AS MLCK was assessed by counting DAPI-stained apoptotic nuclei exhibiting condensed, marginalized chromatin. MLCK inhibition by GFP-AS MLCK overexpression significantly inhibited TNF--induced apoptosis (
77% reduction) (Fig. 2B
). We next explored the effect of EC MLCK inhibition on TNF--induced caspase 3 activation, detected by fluorescence released by caspase 3-specific substrate cleavage in intact cells. TNF--induced caspase 3 activation was inhibited by a reduction in MLCK expression, but not by empty vector controls (Fig. 3
, arrows). Similarly, expression of the dominant/negative EC MLCK-ATPdel construct (Fig. 4
A) produced a marked reduction in the TNF--induced caspase 3 activation compared with untransfected wild-type endothelial cells (Fig. 4B
) or empty vector (not shown). Together, these data indicate that a catalytically intact EC MLCK is required for TNF--induced effector caspase activation and endothelial cell apoptosis. In contrast, compared with untransfected cells, Fig. 4
demonstrates that overexpression of EC MLCK-1745, a constitutively active mutant (unpublished observation), significantly accelerates TNF--induced activation of the executioner caspase 7 and apoptosis accompanied by pronounced cellular condensation and membrane blebbing (Fig. 4C
, panels iii and iv).
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Colocalization of EC MLCK and caspase 8 along actin stress fibers after TNF-
We next explored the relationship between EC MLCK activation and the initiation of the apoptotic sequence by assessing the spatial distribution of caspase 8 and MLCK in endothelial cells overexpressing the GFP-tagged EC MLCK 2 splice variant. Our initial experiments demonstrated that TNF- challenge redistributes caspase 8 staining from a diffuse cytosolic pattern to a linear pattern that mirrors the developing stress fibers formed in response to TNF- activation of the contractile apparatus (Fig. 5
A). This is more clearly depicted in Fig. 5B
, where GFP MLCK 2 expression directly increases stress fiber formation with MLCK sharply aligned along each stress fiber. TNF- challenge produces further increases in stress fiber formation with enhanced MLCK staining in a linear pattern along the stress fibers (Fig. 5C
) in conjunction with prominent alteration in the pattern of caspase 8 localization, with a dramatic linear distribution along the actin cables. Sixty minutes after TNF-, caspase 8 can be found heavily colocalized with GFP-MLCK along actin stress fibers (Fig. 5C
). These results strongly suggest a spatial interaction between the actomyosin contractile apparatus and caspase 8 and provide a potential mechanism by which the cytoskeletal rearrangement potentially participates in the assembly of the TNF- death receptor complex.
Caspase 3 directly cleaves EC MLCK and enhances Ser/Thr kinase activity
Cytoskeletal components are a prominent target of caspase-mediated proteolysis during cellular apoptosis, triggering subsequent morphological changes and inactivating prosurvival pathways (16
, 17)
. We next investigated whether MLCK serves as a biologically relevant substrate for caspase cleavage using in vitro and in vivo assays. Coincubation of recombinant EC MLCK1 or SM MLCK with active recombinant caspase 3, a central effector caspase (10 U/mL or 660 U/µg MLCK protein, 120 min), led to the generation of peptide cleavage products visualized by Coomassie staining of SDS-PAGE gels (shown for EC MLCK in Fig. 6
A) and by Western blot with anti-MLCK antibody (shown for SM MLCK in Fig. 6A
). Caspase 3 inhibitors (Z-DEVD-fmk) inhibited the appearance of cleavage products, indicating that the MLCK proteolysis was caspase specific (Fig. 6)
. TNF--treated endothelial cells previously transfected to overexpress V5 epitope-tagged EC MLCK-1745 also demonstrate caspase-specific EC MLCK cleavage, visualized by immunoblots with anti-V5 antibody (Fig. 6)
. The primary cleavage product exhibited a molecular mass of 65–70 kDa, consistent, with a putative cleavage site we noted at VTVD (D1188) that resides between the actin binding region (#922–1031) and the putative myosin light chain binding region (# 1321–1457), based on sequence identity with the rabbit SM MLCK myosin light chain binding domain (18)
. Cleavage products were absent in TNF--treated endothelial cells overexpressing the V5 epitope-tagged dominant/negative EC MLCK-ATPdel (Fig. 6)
, suggesting either lack of caspase activation in these cells (as suggested by Fig. 4
) or, less likely, the absence of a functional cleavage site within this MLCK mutant. To identify the putative cleavage site(s), we used mass spectroscopy analysis of the cleaved peptides and determined that caspase 3-mediated cleavage of EC MLCK occurs at LKKD (D1703), with secondary cleavage at VNQD (D226), NQDD (D227) or SCKD (D327) in the amino-terminal region of EC MLCK1 (Fig. 6C
). To identify the potential functional consequences of caspase 3 cleavage of MLCK, we measured Ca2+-calmodulin (CaM) -dependent MLCK activity in an in vitro assay performed under conditions similar to those used for in vitro MLCK cleavage by caspase 3. As depicted in Fig. 7
, basal EC MLCK1 enzymatic activity (in the absence of calmodulin) is significantly enhanced by caspase 3 interaction, which appears to proceed in a cleavage-independent manner since the addition of Z-DEVD-fmk (1–10 µM), which effectively inhibited cleavage (Fig. 7A
), did not reduce the caspase 3-enhanced MLCK enzymatic activity (Fig. 7B
). Although we found strong evidence for caspase-dependent cleavage of SM MLCK, caspase–SM MLCK interaction did not increase the enzymatic activity of SM MLCK (Fig. 7)
. Caspase 3-EC MLCK interaction did not further increase kinase activity under conditions where MLCK was maximally activated at baseline, i.e., in the presence of increased Ca2+ and CaM concentrations (Fig. 7
); the removal of Ca2+ and CaM from the reaction mixture abolished the caspase 3-dependent increases in MLCK activity (not shown). Together, these experiments suggest that caspase 3 directly increases EC MLCK1 activity in the presence of Ca2+ in a cleavage-independent and isoform-specific manner, strongly implicating a direct effect of protein–protein interaction.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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on the pulmonary endothelium are diverse and include increases in cellular apoptosis, vascular permeability, and leukocyte diapedesis, each response appearing to involve activation of the MLCK-driven endothelial contractile apparatus (9
, 19
, 20)
. TNF- triggers MLC phosphorylation-dependent endothelial actin cytoskeletal rearrangement with intercellular gaps and stress fiber formation (9)
, where the molecular motor underlying actin cytoskeletal changes is myosin, an ATPase capable of generating mechanical force by promoting translational movement across the actin fibers (21)
. Myosin II is the main nonmuscle class of myosin, consisting of two sets of myosin heavy chains (200 kDa) and two sets of MLC (16–20 kDa), and is regulated by MLC phosphorylation catalyzed by MLCK. Previous reports have investigated the involvement of MLC phosphorylation in apoptotic membrane blebbing (5
, 6)
, a process distinct from the execution phase of apoptosis. The potential involvement of MLCK in the progression to apoptosis was suggested in a TNF--challenged tumor cell line U937 (22)
as well as by our previous work, which, using pharmacological inhibition indicated that activation of the multifunctional MLCK by TNF- is critical for the execution of the programmed cell death (9)
. The data presented in this report provide several lines of evidence that TNF--induced endothelial cell MLC phosphorylation, actin rearrangement, and apoptosis are critically dependent on activation of EC MLCK. Reductions in MLCK activity by either overexpression of antisense MLCK oligonucleotides or a EC MLCK mutant lacking the ATP binding site (EC MLCK-ATPdel) markedly attenuated TNF--induced MLC phosphorylation, stress fiber formation, and cellular apoptosis, whereas overexpression of a constitutively active EC MLCK lacking the autoinhibitory domains (EC MLCK-1745), resulted in increased actin microfilament rearrangement and increases in programmed cell death. These data strongly implicate MLCK as an essential mediator of TNF--induced actin rearrangement and apoptosis in endothelial cells. The mechanisms by which MLCK modulates apoptosis are not entirely understood.
Jin et al. reported in an immortalized epithelial cell line (MDCK) that MLCK-dependent actin rearrangement facilitates the recruitment of the TNFR1 to the plasma membrane (8)
. Equally plausible is EC MLCK involvement in initiator caspase activation via the actomyosin contractile rearrangement, which is critical to the assembly of the TNF- death receptor complex. Apoptotic TNF- signals are transmitted through TNFR1, which, through adaptor proteins, activates procaspase 8 (via proximity-induced oligomerization) and subsequently the caspase cascade (caspases 3 and 7). These signals further inhibit the NF-
B-mediated anti-apoptotic pathways, and result in execution of the apoptotic program and typical morphologic changes of cellular shrinkage and apoptotic body formation. Two protein kinases with significant homology to EC MLCK, ZIP kinase and DAP kinase, exhibit discrete catalytic, calmodulin binding, ankyrin repeat, and death domain regions. The exact role of DAP kinase in programmed cell death is not clear as DAP kinase has been reported to promote (23)
as well as inhibit apoptosis (24)
. It is also unclear whether the capacity for DAP kinase to exert apoptotic regulatory functions is related to its ability to phosphorylate MLC or, through its interaction with other molecules, via the death domain. A potential cytoskeletal mechanism of caspase activation has been suggested, with recruitment of "death effector filaments" by death effector domain-containing proteins, triggering caspase 8 recruitment and initiating apoptosis (25)
. Siegel et al. propose that recruitment to these filaments is highly efficient and with a dramatic increase in the local concentration of procaspases, thereby facilitating caspase activation (25)
. Although EC MLCK does not contain either an ankyrin-repeat region or a death domain, our results not only confirm our prior observation that that MLCK activity is important for TNF--induced caspase 8 activation in endothelial cells (9)
, but provide the novel observation that colocalization and stable association between overexpressed MLCK and caspase 8 occur along actin stress fibers, consistent with a defined spatial interaction and early involvement of MLCK in the progression to cell death.
These results imply a critical role for MLCK in apoptotic signaling either through its role as an actin rearrangement modulator or through an as yet unknown direct interaction with the apoptotic molecular machinery. It is increasingly recognized that cytoskeletal components are substrates for the caspase cleavage with morphologic and functional consequences (16)
. For example, it was recently demonstrated that Rho kinase is a substrate for caspase 3, with the cleaved fragment exhibiting a constitutively active kinase activity resulting in increased membrane blebbing in Jurkat cells (6)
. Using complementary in vivo and in vitro approaches, we investigated whether EC MLCK is also a specific substrate for the apoptotic caspase cascade compared with the SM MLCK isoform. The high molecular mass EC MLCK isoform (210–214 kDa) and its SM MLCK counterpart (130-150 kDa) share identical actin binding, MLC binding, catalytic, and Ca2+/CaM regulatory domains. The extreme carboxyl-terminal kinase-related protein (KRP) domain, which binds myosin, is identical within EC MLCK and SM MLCK isoforms, but can also be expressed as an independent protein capable of stabilizing myofilaments in vitro (7)
. However, the function of the 922-amino acid amino terminus, unique to the high molecular weight EC MLCK isoform, is largely unknown with the exception of its involvement in the post-translational modification of EC MLCK by p60src (7)
. We found that both EC MLCK and SM MLCK are specific and direct substrates of caspase 3 cleavage, suggesting that cleavage occurs in the common carboxyl-terminal portion of the molecule. Our in vivo studies using endothelium transfected with EC MLCK-1745 demonstrated that EC MLCK-1745 is cleaved by TNF--activated endogenous caspases, with VTVD (D1188) a strong candidate site, based on structure similarities with a known caspase cleavage site, VEVD, present within cytokeratin 18 and lamin B1 (16)
. Endothelium similarly transfected to overexpress the dominant/negative EC MLCK-ATPdel construct failed to generate TNF--induced cleavage products, consistent with our results that MLCK is required for caspase activation. Sequence analysis within the EC MLCK-ATPdel revealed identical sequence homology with the known caspase cleavage sites present in EC MLCK-1745 and EC MLCK, making the alternative explanation that the EC MLCK-ATPdel mutant lacks the specific functional caspase cleavage site less likely. Mass spectroscopy analysis of EC MLCK cleavage fragments from in vitro experiments indicated a primary cleavage site at LKKD (D1703) and additional secondary cleavage sites within the unique amino terminus at VNQD (D226), NQDD (D227), and SCKD (D327), similar to those described (XXQD and XXDD) within Bax, presenilin, PKC
, and calpostatin, respectively (16)
. Additional studies using site-directed mutagenesis will be needed to detect with precision the primary caspase 3 cleavage site on EC MLCK in vivo.
In addition to the novel characterization of EC MLCK as a caspase target, we observed enhanced CaM-independent enzymatic activity after caspase 3 interaction with EC MLCK. The generation of a constitutively active fragment is consistent with cleavage within the carboxyl terminus region (between the catalytic and the autoinhibitory domains) and is not unprecedented, as Rho kinase was also found to be a caspase 3 substrate that, when cleaved, assumes a constitutively active function, resulting in enhanced apoptotic membrane blebbing (6)
. We did not anticipate, however, the lack of modulation by the caspase inhibitor Z-DEVD-fmk of the caspase3-induced enhanced kinase activity. These results appear to suggest a proteolytic-independent phenomenon, where increases in kinase activity occur as a direct result of caspase 3 binding to EC MLCK. Despite significant cleavage of SM MLCK by caspase 3, we did not observe enhanced SM MLCK activity when exposed to caspase 3 under similar conditions. Together, these results suggest a direct and novel interaction of caspase 3 with the unique amino terminus of the EC MLCK isoform that results in an enhanced MLCK enzymatic activity, which does not require proteolytic cleavage. Future work will explore whether the interaction between active caspase 3 and MLCK is necessary for endothelial cell apoptosis.
In summary, we have examined the role of the MLCK-dependent actomyosin microfilament modulation of TNF--induced endothelial cell apoptosis. Our results strongly confirm MLCK as a critical participant in TNF- induced apoptosis signaling in endothelial cells, leading to initiator and effector caspase activation. The exact mechanisms by which the MLCK-mediated cytoskeletal changes modulate the apoptotic process remain to be demonstrated, but our results suggest direct involvement in upstream adaptor protein recruitment and caspase 8 activation by the TNFR1 death domains. EC MLCK appears to serve as a substrate for caspase 3 cleavage with direct interaction leading to increased MLCK enzymatic activity in a cleavage-independent manner. Future studies will determine the exact sites of EC MLCK cleavage in vivo and the functional consequences of caspase 3-MLCK interaction. Elucidation as to how cytoskeletal machinery participates in the cell’s vital decisions for survival or cell death may provide clues about the complex molecular events involved in regulation of endothelial cell apoptosis.
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ACKNOWLEDGMENTS |
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Received for publication August 2, 2002.
Revision received November 21, 2002.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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