Apoptotic death of vascular smooth muscle cells (SMCs) is a prominent feature of blood vessel remodeling and various vascular diseases. We have previously shown that protein kinase C-δ (PKC-δ) plays a critical role in SMC apoptosis. In this study, we tested the importance of PKC-δ proteolytic cleavage and tyrosine phosphorylation within the apoptosis pathway. Using hydrogen peroxide as a paradigm for oxidative stress, we showed that proteolytic cleavage of PKC-δ occurred in SMCs that underwent apoptosis, while tyrosine phosphorylation was detected only in necrotic cells. Furthermore, using a peptide (z-DIPD-fmk) that mimics the caspase-3 binding motif within the linker region of PKC-δ, we were able to prevent the cleavage of PKC-δ, as well as apoptosis. Inhibition of PKC-δ with rottlerin or small-interfering RNA diminished caspase-3 cleavage, caspase-3 activity, cleavage of poly (ADP-ribose) polymerase, cleavage of PKC-δ, and DNA fragmentation, confirming the previously reported role of PKC-δ in initiation of apoptosis. In contrast, z-DIPD-fmk markedly diminished caspase-3 activity, cleavage of PKC-δ, and DNA fragmentation without affecting cleavage of caspase-3 and poly (ADP-ribose) polymerase. Taken together, our data suggest that caspase-3-mediated PKC-δ cleavage underlies SMC apoptosis induced by oxidative stress, and that PKC-δ acts both upstream and downstream of caspase-3.
- oxidative stress
programmed cell death or apoptosis of vascular smooth muscle cells (SMCs) plays a vital role in normal development of the circulatory system, as well as in arterial remodeling during pathogenesis of vascular diseases, including atherosclerosis, restenosis, and abdominal aortic aneurysms. It has been demonstrated in experimental models that deregulation of the apoptotic pathway within vascular SMCs disrupts the balance between proliferation and apoptosis, which determines the size of neointimal lesions (32, 37). Recently, it has been reported that apoptosis of vascular SMCs alone is sufficient to induce features of plaque vulnerability in the apolipoprotein E-deficient atherosclerotic model in mice (6).
Apoptosis can be induced by various stimuli that elicit different signaling pathways, but the ultimate initiation and execution of apoptosis is mediated by a common cascade involving caspases, a structurally related group of cysteine proteases. Caspases are synthesized as inactive zymogens, and the activation of the caspase cascade involves proteolysis and activation of initiation caspases that, in turn, cleave and activate the execution caspases (35). While apoptosis of all mammalian cells shares this central pathway, the upstream mediators vary, depending on the nature of stimuli, and may be cell-type specific.
PKC is a family of major serine-threonine kinases that shares a similar structure, including an NH2-terminal regulatory domain and a COOH-terminal catalytic domain connected via a hinge region. One of the novel PKC isoforms, protein kinase C-δ (PKC-δ), has been shown to be associated with the response to DNA damage and other apoptotic stimuli in specific cell types. In the past, our laboratory and others have reported that activation of PKC-δ is a critical component in the apoptotic response of vascular SMCs to oxidative stress (22, 33). In particular, PKC-δ null mice were found to show diminished apoptotic responses in a vein graft bypass model (22). However, in addition to being proapoptotic, PKC-δ is also known to mediate other cellular events of vascular SMCs in response to nonapoptotic stimuli, including cell proliferation, attachment, and production of matrix proteins (14, 17, 23, 26). It remains unclear why activation of PKC-δ only leads to cell apoptosis under certain circumstances.
Several activation mechanisms for PKC-δ are known: activation by diacylglycerol after serine and threonine phosphorylation at the motif sites, formation of the active enzyme by tyrosine phosphorylation, generation of the catalytic fragment by proteolytic cleavage, and PKC-δ-PKC-δ interaction. Of these, tyrosine phosphorylation and cleavage by caspase-3 have been shown to be important for the proapoptotic role of PKC-δ under oxidative stress in nonvascular cell types (19, 24). PKC-δ is shown to undergo cleavage in response to etoposide in glioma and acinar cells (3, 31), UV radiation in keratinocyes (9), tumor necrosis factor-related apoptosis inducing ligand in glioma cells (27), cisplatin in glioma and small cell lung cancer (2, 29), hydrogen peroxide (H2O2) in neuronal cells (19), and mitomycin C in gastric adenocarcinoma (28), and its cleaved catalytic fragment has been associated with the apoptotic function of PKC-δ (11, 30). On the other hand, tyrosine phosphorylation at positions 311, 332, and 512 have been identified to become phosphorylated following exposure to H2O2 in COS-7 cells (21), and the enzyme recovered to be constitutively active. However, the functional consequence of these two activated forms of PKC-δ is still controversial.
In our present study, we tested in vascular SMCs whether PKC-δ undergoes cleavage and tyrosine phosphorylation when cells are stressed by H2O2. Furthermore, by using inhibitors or small-interfering RNAs (siRNAs), we compared the effects of inhibition of PKC-δ and PKC-δ cleavage on key steps within the apoptotic pathway. We found that cleavage of PKC-δ plays a similar role in mediating SMC apoptosis as reported in other cell types; however, the involvement of PKC-δ tyrosine phosphorylation appears to be cell type specific.
MATERIALS AND METHODS
Rottlerin, dimethyl sulfoxide (DMSO), and H2O2 were purchased from Sigma (St. Louis, MO), z-DEVD-fmk (fluoromethyl ketone) from Calbiochem (La Jolla, CA), and Q-VD-OPh from R&D Systems (Minneapolis, MI). The tetrapeptide Z-Asp(OMe)-Ile-Pro-Asp(OMe)-FMK or z-DIPD-fmk is based on the sequence of rat PKC-δ (position 324–327). Z-DIPD-fmk, structurally similar to the commercial caspase-3 inhibitor z-DEVD-fmk, was synthesized at the proteomics resource center of Rockefeller University using standard solid-phase peptide synthesis based on the description previously reported (18). FMK-derivatized peptides are designed for use as irreversible caspase inhibitors with no added cytotoxic effects. This particular inhibitor also contains a benzyloxycarbonyl group (designated “Z”) at the NH2-terminus and O-methyl side chains for enhanced cellular permeability.
Rat aortic SMC line (A10 cells), obtained from American Type Culture Collection, was grown as recommended at 37°C in 5% CO2 in Dulbecco's minimal essential medium (Invitrogen, Grand Island, NY), modified to contain 4 mM l-glutamine, 4.5 g/l glucose, 1 mM sodium pyruvate, 1.5 g/l sodium bicarbonate, supplemented with 10% fetal bovine serum (Gemini, Woodland, CA) and antibiotics. DNA transfection was carried out using a nueclofection kit from Lonza Cologne AG (Cologne, Germany). Plasmid vectors that encode the wild-type PKC-δ or mutants in which tyrosine residues 311 or 322 were replaced with phenylalanine were kindly provided by Dr. Kikkawa (16).
A10 cells were made quiescent by incubation in medium containing 0.5% fetal bovine serum for 48 h and then treated with H2O2 at various concentrations and times. Cells were lysed in 50 mM Tris·HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, and 0.1% SDS, and subjected to SDS-PAGE and transferred to a polyvinylidene difluoride (Bio-Rad, Hercules, CA) membrane. Membranes were incubated with rabbit polyclonal antibodies to the NH2-terminus of PKC-δ or phosphorylated tyrosine 311 (p-Tyr311) (Santa Cruz Biotechnology, Santa Cruz, CA), poly (ADP-ribose) polymerase (PARP), caspase-3, or cleaved caspase-3 (Cell Signaling Technology, Beverly, MA) and β-actin (Sigma). Labeled proteins were visualized with an enhanced chemiluminescence system (PerkinElmer Life Sciences, Boston, MA).
Immunoprecipitation was carried out as described previously (15). Briefly, cells were lysed in Nonidet P-40 buffer. Total protein concentration was determined by a modification of the method of Lowry, and the protein amount of each sample was equalized. Protein A-Sepharose beads (Santa Cruz Biotechnology) and 5 μg of antibody specific to phosphorylated tyrosine (Santa Cruz) were added to lysates and incubated at 4°C with constant rotation overnight. Following centrifugation, pellets were washed five times with Nonidet P-40 buffer and one time with 50 mM Tris. The final pellet was resuspended in 8 μl of sample buffer and heated to 100°C for 5 min, and samples were subjected to SDS-PAGE.
DNA fragmentation was determined using the Cell Death Detection ELISA system (Roche Applied Science, Indianapolis, IN), an assay based on a quantitative sandwich-enzyme-immunoassay principle using mouse monoclonal antibodies directed against DNA and histones. Cellular extracts were incubated in 96-well plates coated with anti-histone antibodies. Plates were then incubated with anti-DNA antibodies conjugated to peroxidase for 2 h, and absorbance was measured at 405 nm.
Activation of PKC was assessed by a nonradioactive Protein Kinase Assay kit (Calbiochem). Cellular extracts were incubated in 96-well plates precoated with peptide pseudosubstrate for PKCs. Phosphorylated substrates were recognized by biotinylated monoclonal antibodies and then detected with horseradish peroxidase-conjugated streptavidin, and abosorbance was read at 492 nm. Activation of caspase-3 was quantified by Western blotting using a polyclonal rabbit antibody that detects the large fragment (17/19 kDa) of activated caspase-3, resulting from cleavage adjacent to Asp175, and Caspase-Glo 3/7 Assay (Promega, Madison, WI) was employed according to the manufacture's instructions. Caspase-3/7 activity was represented by measuring release of amino-luciferin as a result of active caspase-3/7 cleaving luminogenic substrate containing the DEVD sequence.
Stealth RNA interference (RNAi) for the PKC-δ sequence, CCG UUC CUG CGC AUC UCC UUC AAU, and for the caspase-3 sequence, ACU ACU GCC GGA GUC UGA CUG GAAA, was purchased from Invitrogen. Cells were transfected with 10 nM siRNA using lipofectamine RNAiMAX (Invitrogen), according to the manufacturer's protocol. Following transfection for 48 h, cells were collected for analysis. Stealth RNAi negative control was employed for the control transfection.
After specified treatment, cells were collected, resuspended in Annexin-V-FLUOS labeling solution (Roche Applied Science), and analyzed on a flow cytometer using 488-nm excitation and a 515-nm band-pass filter for fluorescein detection and a filter >600 nm for propidium iodide (PI) detection.
Values were expressed as a fold increase (means ± SE) of at least three independent experiments. Paired data were evaluated by Student's t-test, and a one-way ANOVA was used for multiple comparisons. Values of P < 0.05 were considered significant.
H2O2 induces dose-responsive activation of caspase-3 pathway and PKC-δ in vascular SMCs.
We began our study by testing for concentrations of H2O2 that were optimal for inducing apoptosis in vascular SMCs. A10 cells were treated with H2O2 at concentrations of 50 μM to 2 mM for 4 h. Doses of H2O2 between 100 and 500 μM caused increases in DNA fragmentation, whereas higher concentrations displayed a decrease (Fig. 1A). Flow cytometric analysis revealed a significantly higher number of apoptotic cells (annexin V+/PI−) after treatment with 400 μM H2O2, and necrotic cells (annexin V+/PI+) after treatment with 2 mM H2O2 (Fig. 1B). Increase of caspase-3 activation, assessed by its cleavage via Western blot (Fig. 1C) and its ability to cleave the DEVD sequence (Fig. 1D), resembled the dose response of apoptosis. PARP, a known caspase-3 substrate and potent regulator of apoptosis, was also cleaved at similar doses (Fig. 1C). Further administration of H2O2 (>800 μM) induced necrosis, accompanied by lesser activation of caspase-3. PKC-δ activity, defined by the difference between the total PKC activity and PKC activity with rottlerin treatment, was significantly increased at 200–400 μM H2O2 and slightly increased at 1–2 mM H2O2 (Fig. 1E). Alternative to rottlerin, we inhibited endogenous PKC-δ by transfecting A10 SMCs with an siRNA specific to PKC-δ, followed by stimulation with 400 μM H2O2. Consistently, a PKC-δ-specific activity was observed in H2O2-treated cells (Supplemental Fig. 1; the online version of this paper contains supplemental data).
H2O2-induced apoptosis is accompanied by proteolytic cleavage, but not phosphorylation of PKC-δ.
We next evaluated how PKC-δ and its proteolytic cleavage relate with SMC response to oxidative stress. Again, A10 SMCs were treated with H2O2 at concentrations of 50 μM to 2 mM for 4 h. Although levels of full-length PKC-δ did not change significantly with H2O2 treatment, the amounts of catalytic fragment resulting from PKC-δ cleavage increased in cells treated with H2O2, ranging from 50 to 500 μM (Fig. 2A). Phosphorylation of PKC-δ was also evaluated through Western blot with the use of a specific antibody for phosphorylated Tyr311. However, phosphorylation at Tyr311 only became positive with 2 mM H2O2 treatment (Fig. 2A). Next, we examined tyrosine phosphorylation of PKC-δ using an alternative method. Following treatment with 500 μM to 3 mM of H2O2 for 4 h, cell lysates were collected and immunoprecipitated with antiphosphorylated tyrosine antibody and then blotted using an anti-PKC-δ antibody. Results showed a robust increase of PKC-δ phosphorylation in cells treated with >1.5 mM H2O2 (Fig. 2A, top). Immunoblot with phosphorylated Tyr311 displayed similar results (Fig. 2B, middle).
Tyrosine phosphorylation within the linker region of PKC-δ has been previously implicated in regulation of PKC-δ cleavage in neuronal cells (19). To test whether a similar mechanism exists in vascular SMCs, we ectopically expressed a PKC-δ mutant baring a Tyr-to-Phe mutation at residue 311 or 322 (Tyr311 or Tyr322). Compared with transfection of a nonspecific cDNA vector (green fluorescent protein), transfection of A10 SMCs with the wild-type PKC-δ cDNA led to an increased level of catalytic fragment accompanied by a similar increase in the full-length PKC-δ molecule (Supplemental Fig. 2). A similar effect on the level of catalytic fragment was also produced by transfection of PKC-δ tyrosine mutants (Supplemental Fig. 2).
To ensure that the lack of PKC-δ cleavage was not caused by a time difference of the reactions, we performed a time course study. In the group treated with 400 μM H2O2, the induction of cleavage began after 2 h and became prominent after 4 h (Fig. 2C), but phosphorylation was not detected at any time point (data not shown). Apoptosis, measured as the amount of DNA fragmentation quantified by ELISA (Fig. 2C) and caspase-3 activity (Fig. 2E), became significant after 4 h. In contrast, the group treated with 2 mM H2O2 showed a transient tyrosine phosphorylation of PKC-δ (data not shown), but no evidence of PKC-δ cleavage nor DNA fragmentation was found at any time point tested (Fig. 2D).
Proteolytic cleavage of PKC-δ is mediated by caspase-3.
To test whether the cleavage of PKC-δ is caspase dependent, we examined the effect of the pan-caspase inhibitor, Q-VD-OPh. Treatment with 20 μM Q-VD-OPh significantly diminished H2O2-induced cleavage of PKC-δ (Fig. 3A, left). Similarly, H2O2-induced apoptosis was almost completely abolished (Fig. 3B, left). The caspase-3 inhibitor, z-DEVD-fmk (50 μM) also inhibited the H2O2-induced PKC-δ cleavage and apoptosis (Fig. 3, A and B, right). However, the effect was only partial compared with the pan-caspase inhibitor. As z-DEVD-fmk may cross react with other effector caspases, we further verified the role of caspase-3 in the proteolytic cleavage of PKC-δ by inhibiting caspase-3 expression in A10 cells with an siRNA. Results showed complete blockage of PKC-δ and PARP cleavage, accompanied by inhibition of apoptosis (Fig. 3, C and D).
Proteolytic cleavage of PKC-δ is necessary for apoptosis.
To further prove that the caspase-mediated cleavage of PKC-δ is an integral step of the apoptotic pathway within SMCs, we synthesized a cell-permeable peptide z-DIPD-fmk that mimics the putative caspase-3 cleavage site within the linker region of rat PKC-δ (Fig. 4A). Administration of 50–100 μM of z-DIPD-fmk blocked cleavage of PKC-δ in SMCs that were treated with 400 μM H2O2 (Fig. 4B). Using a caspase-3-specific substrate, we showed that z-DIPD-fmk diminished the activity of caspase-3 elicited by H2O2 (Supplemental Fig. 3). However, the same peptide did not decrease the level of cleaved caspase-3, suggesting it has a minimal effect on caspase-9 (Fig. 4B). Importantly, z-DIPD-fmk-treated cells became resistant to apoptosis, as demonstrated by the diminished DNA fragmentation (Fig. 4C). Flow cytometric analysis confirmed that the decrease of DNA fragmentation was accompanied by the decrease of apoptosis, and not by the increase of necrosis (Fig. 4D).
PKC-δ is also necessary for the activation of caspase-3.
Since vascular SMCs isolated from PKC-δ null mice show defective caspase-3 activation, as well as DNA fragmentation, it is thought that PKC-δ plays a role in the apoptotic pathway upstream of the initiation of the caspases cascade (22, 33). However, our proceeding experiments showed that inhibition of PKC-δ cleavage with the z-DIPD-fmk peptide diminished DNA fragmentation without affecting caspase-3 cleavage/activation. Therefore, we sought to inhibit PKC-δ with a more general approach by employing its chemical inhibitor rottlerin or siRNA. Administration of 2 μM rottlerin before H2O2 treatment attenuated both caspase-3 activation and apoptosis (Fig. 5). Moreover, silencing PKC-δ gene expression with an siRNA showed a similar broad suppression of the apoptotic pathway: diminished cleavage of caspase-3 and its substrate PARP and PKC-δ, activity of caspase-3/7, as well as DNA fragmentation (Fig. 6).
Results from this study demonstrated not only the prominent presence of the catalytic fragment in vascular SMCs that were challenged with apoptotic stimuli, but also the pivotal role of this proteolysis in apoptosis. In contrast, tyrosine phosphorylation of PKC-δ seemed to be related with necrosis. Moreover, inhibitors, such as the pan-caspase inhibitor Q-VD-OPh and the caspase-3 inhibitor z-DEVD-fmk, attenuated the level of catalytic fragment detected in H2O2-treated SMCs. The potency of these caspase inhibitors to block PKC-δ cleavage paralleled their ability to inhibit cell apoptosis. Most importantly, the peptide inhibitor z-DIPD-fmk, designed to specifically mimic the caspase-3 cleavage site within PKC-δ inhibited both PKC-δ cleavage and apoptosis.
In the past, a report on Chinese hamster ovaries cell line overproducing PKC-δ has shown that H2O2-induced apoptosis was enhanced compared with that overproducing wild type, but no formation of the catalytic domain could be observed (20). However, our findings that suggest the caspase-mediated PKC-δ cleavage as an important apoptotic signaling step is consistent with other reports, such as the one in dopaminergic cells showing caspase-3 cleaves PKC-δ at 324DIPD327 to generate a 38-kDa regulatory domain and a 41-kDa constitutively active fragment (4, 12, 13, 19) in response to DNA-damaging agents. Mutations in the caspase cleavage site have also been shown to inhibit both the pro- and antiapoptotic effects of PKC-δ (8, 10, 27), supporting a role of PKC-δ cleavage in both cell apoptosis and survival.
PKC-δ is the most extensively tyrosine-phosphorylated isoform among the PKC family, and its phosphorylation is thought to be essential for the activation (7, 10, 34) and localization (38) within the cell. In neuronal cells, phosphorylation on Tyr311 was postulated as the mechanism that promotes the cleavage of PKC-δ. We did not detect any significant level of phosphorylation on Tyr322 in apoptotic SMCs. Although Tyr311 became phosphorylated in SMCs treated with high concentrations of H2O2, flowcytometric analysis indicated that, under these conditions, SMCs were necrotic rather than apoptotic. Furthermore, mutating tyrosine on residue 311 or 322 did not antagonize H2O2-induced cleavage of PKC-δ. In fact, both tyrosine mutants were able to undergo cleavage in a similar fashion to that of the wild-type PKC-δ in SMCs. We postulate that the regulatory mechanism underlying PKC-δ cleavage is cell type specific. In vascular SMCs, tyrosine phosphorylation of PKC-δ may not be a major triggering event in the apoptotic in response to H2O2.
Based on the different effects on caspase cascade produced by different PKC-δ inhibitory strategies, we propose a model to explain the dual functions of PKC-δ in SMC apoptosis (Fig. 7). Inhibition of PKC-δ by rottlerin or silencing gene expression by siRNA attenuated PKC-δ functions, both upstream and downstream of caspase-3, thus blocking both initiation (measured by caspase activation) and execution (measured by DNA fragmentation) phases of apoptosis. Our postulated upstream function of PKC-δ is consistent with early reports that showed PKC-δ translocation to the mitochondria, cytochrome c, and successive activation of the caspase cascade (25). Furthermore, the fact that the specific inhibition of PKC-δ cleavage by z-DIPD-fmk blocked intranucleosomal cleavage of DNA (measured by the ELISA assay) and translocation of phosphatidylserine from the inner part of the plasma membrane to the outer layer (measured by annexin V stain) without affecting caspase activation or PARP cleavage suggests that the cleaved PKC-δ product is a key signaling component for the execution of the death pathway. Potential targets for cleaved PKC-δ have been reported to be p53, Rad9, topoisomerase IIα, lamin-β, p73β, and DNA-PK (1, 7, 30, 39–41), but this still needs to be studied in vascular SMCs. Although we do not have a definite explanation for z-DIPD-fmk to lack effect on PARP, the result was consistent with the effect on neuronal cells (18) and may be associated with the differences in affinity of the substrates. Of note, it is thought that cleavage of PARP executes apoptosis by conserving NAD+ and ATP (36) or by its native form losing its key homeostatic function as a DNA repair enzyme (5). The fact that z-DIPD-fmk inhibited intranucleosomal cleavage of DNA and translocation of phosphatidylserine without affecting the cleavage of PARP suggests PARP itself is not sufficient for apoptosis.
A major limitation of this present study is the use of an aortic SMC cell line. The fact that A10 SMCs display the same dependency on PKC-δ for the regulation of apoptosis as what has been reported in vivo (22) suggests the mechanistic pathway obtained from the present study is likely to be applicable to physiological and pathological states in animals.
In summary, our results demonstrate that the proteolytic cleavage of PKC-δ plays a pivotal role in the signal transduction pathway leading to vascular SMC apoptosis. Given the ubiquitous nature of PKC-δ expression and its involvement in regulation of a wide range of cellular functions, a strategy that specifically targets PKC-δ cleavage may provide an efficient yet safe approach to block cell apoptosis of vascular SMCs under oxidative stress.
This work was supported by Public Health Service Grant R01 HL-81424 (K.C. Kent and B. Liu) from the National Heart, Lung, and Blood Institute and American Heart Association Grant-in-aid 0455859T (B. Liu).
No conflicts of interest are declared by the author(s).
The authors thank Sophia Chu and Chunjie Wang for technical assistance, Stephanie Morgan for editorial revisions, Dr. Ushio Kikkawa (Biosignal Research Center, Kobe University, Japan) for providing mutant plasmids and for advice and suggestions, and Drs. Haiteng Deng and Nagarajan Chandramouli (Rockefeller University, New York, NY) for the synthesis of z-DIPD-fmk.
Current address of K. Kamiya: Second Department of Surgery, Yamanashi University, Shimokatoh, Chuo, Yamanashi 409-3898.
- Copyright © 2009 the American Physiological Society