Proteome Analysis and Functional Expression Identify Mortalin as an Antiapoptotic Gene Induced by Elevation of [Na+]i/[K+]i Ratio in Cultured Vascular Smooth Muscle Cells
Apoptosis of vascular smooth muscle cells (VSMCs) plays an important role in remodeling of vessel walls, one of the major determinants of long-term blood pressure elevation and an independent risk factor for cardiovascular morbidity and mortality. Recently, we have found that apoptosis in cultured VSMCs can be inhibited by inversion of the intracellular [Na+]/[K+] ratio after the sustained blockage of the Na+,K+-ATPase by ouabain. To understand the mechanism of ouabain action, we analyzed subsets of hydrophilic and hydrophobic VSMC proteins from control and ouabain-treated cells by 2-dimensional electrophoresis. Ouabain treatment led to overexpression of numerous soluble and hydrophobic cellular proteins. Among proteins that showed the highest level of ouabain-induced expression, we identified mortalin (also known as GRP75 or PBP-74), a member of the heat shock protein 70 (HSP70) superfamily and a marker for cellular mortal and immortal phenotypes. Northern and Western blotting and immunocytochemistry all have confirmed that treatment of VSMCs with ouabain results in potent induction of mortalin expression. Transient transfection of cells with mortalin cDNA led to at least a 6-hour delay in the development of apoptosis after serum deprivation. The expression of tumor suppressor gene, p53, in mortalin-transfected cells was delayed to the same extent, and the expressed protein showed abnormal perinuclear distribution, suggesting that p53 is retained and inactivated by mortalin. Our studies therefore define a new [Na+]i/[K+]i-responsive signaling pathway that may play an important role in the regulation of programmed cell death in VSMCs.
Remodeling of the blood vessel plays an important role in a variety of human vascular disorders, including hypertension,1–3⇓⇓ atherosclerosis,4 arterial injury, and restenosis after angioplasty.5–7⇓⇓ Apoptosis (programmed cell death) of vascular smooth muscle cells (VSMCs) has recently been identified as the main factor contributing to the regulation of their number during remodeling,8–12⇓⇓⇓⇓ which inspired numerous studies of the mechanisms of the induction and progression of VSMC apoptosis. The execution phase of apoptosis in VSMCs is triggered similarly to that in the other cell types by activation of the caspase cascade, cleavage of intracellular proteins, and final disintegration of the cell. In contrast, the induction phase is specific for different subtypes of remodeling and involves the integration of multiple pro- and antiapoptotic signals, including the expression of death receptors, protooncogenes, and tumor suppressor genes.13–19⇓⇓⇓⇓⇓⇓
Our recent studies showed that inhibition of the VSMC Na+,K+ pump with ouabain, or in K+-free medium, rescues cells from apoptosis triggered by a number of factors including serum deprivation.20 Equimolar substitution of extracellular Na+ with K+ completely abolished the effect of ouabain20 showing that antiapoptotic action was indeed caused by the inversion of [Na+]i/[K+]i ratio. The development of cell death was blocked upstream of caspase-3 activation20 and was cell specific.20–24⇓⇓⇓⇓
The mechanism of the observed phenomenon most probably involves upregulation of antiapoptotic proteins induced by the elevation of [Na+]i/[K+]i ratio because ouabain significantly induced DNA and RNA synthesis in treated VSMCs,25,26⇓ whereas inhibitors of RNA and protein synthesis completely abolished antiapoptotic action of ouabain.25 In this study, we analyzed the VSMC proteome to identify proteins induced after the inhibition of the Na+,K+ pump. Among identified gene products, a heat shock protein 70 (HSP70) family protein, mortalin, was shown to inhibit VSMC apoptosis via inactivation of the proapoptotic tumor suppressor gene, p53.
Materials and Methods
VSMCs from the aorta of male Brown-Norway (BN.lx) rats and VSMC-E1A cells stably transfected with E1A adenoviral protein were obtained as described27,28⇓ and cultured in Dulbecco’s modified Eagle medium with 10% calf serum (CS). BN.lx rats were obtained from the Institute of Physiology, Academy of Science Czech Republic, and treated in accordance with the procedures outlined in the Guide for the Care and Use of Experimental Animals, endorsed by the Canadian Institutes for Health Research.
Cells were extracted with 40 mmol/L Tris base buffer pH 9.5 and then with 8 mol/L urea, 4% CHAPS, and 2 mmol/L tributylphosphine (TBP).29 Extracts were loaded by rehydration on nonlinear immobilized pH gradient 18-cm strips 3–10 (Pharmacia).
Two-dimensional electrophoretic (2DE) was performed as described previously.29 Gels were stained with Coomassie Brilliant Blue, scanned, and analyzed using PDQuest software (Bio-Rad). To create the composite gels, 5 gels from similar samples were aligned and the relative intensities of matched individual protein spots were averaged. Proteins that showed at least 2-fold difference in intensity between composite gels of control and ouabain-treated cells and whose expression level was changed on all individual gels were excised for identification.
Gel pieces containing selected protein spots were treated overnight with trypsin (Promega). Peptide fragments were extracted and analyzed by mass spectrometry.
Nano–LC-MS-MS experiments were conducted on a Q-TOF 2 hybrid quadruple/time-of-flight equipped with a CapLC (Micromass) liquid chromatograph. Chromatographic separations were achieved on a Pepmap C18 precolumn (LC Packings). Protein identification was conducted using Mascot (Matrix Science) with an NCBI protein database.
Immunofluorescence Microscopy of VSMCs
Cells were treated with MitoTracker MitoFluor Red 594 (Molecular Probes), fixed with 3% paraformaldehyde, permeabilized by 0.3% Triton X-100, and stained with mouse monoclonal anti-mortalin antibodies or anti-p53 antibodies, and counterstained with Oregon green 488–conjugated anti-mouse IgG antibodies. Finally, cells were stained with DAPI. Slides were studied on a Zeiss LSM510 inverted confocal microscope (Carl Zeiss Inc) or Nikon Eclipse E6000 direct epifluorescence microscope.
Western and Northern Blotting
VSMC lysates were resolved by SDS-PAGE and electrotransferred to PVDF membrane. The detection was performed with monoclonal anti-human mortalin antibodies or anti-human p53 antibodies.
Ten micrograms of total VSMC RNA was separated by a 1% agarose-formaldehyde gel electrophoresis and blotted onto Hybond-N+ membrane. Mortalin and β-actin cDNA probes were labeled with 32P-dCTP using a multiprime DNA labeling kit (Amersham-Pharmacia Biotech).
Expression of Mortalin in VSMC-E1A Cells
Mammalian expression vector pCMV-SPORT-Mot containing human mortalin cDNA was obtained from Research Genetics. VSMCs were transiently transfected with pCMV-SPORT-Mot and pIRES2-EGFP-Mot expression vectors using Effectene reagent (Qiagen). pCMV-SPORT and pIRES2-EGFP vectors were used for mock transfection.
An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.
Proteome Changes in Ouabain-Treated Cells
To identify VSMC proteins whose expression was significantly changed by ouabain, we compared 2DE maps of VSMC proteome. To increase sensitivity, we performed a sequential extraction of VMSC proteins first with buffer solution to extract all soluble proteins (≈40% to 50% of total cell protein) and then with detergent to extract hydrophobic cellular proteins (≈40% of protein). Altogether, ≈370 protein spots were detected in buffer extracts and ≈260 protein spots in detergent exacts. To create the composite gels (Figure 1), 5 gels from similar samples were aligned and the relative intensities of matched individual protein spots were averaged. The most significant changes in response to ouabain treatment were detected in the pool of soluble proteins (Figure 1A) (142 proteins induced, 56 proteins suppressed), whereas only 16 hydrophobic proteins were induced and 48 suppressed in ouabain-treated VSMCs (Figure 1B). For identification, we selected proteins that showed at least 2-fold difference in intensity on composite gels between control and ouabain-treated cells and whose expression level was changed on all individual gels in the same series of gels (Figure 1).
Twenty proteins whose expression was induced or suppressed in ouabain-treated cells were positively identified with at least 3 MS-MS spectra by nano–LC-MS-MS (Table). Most of the proteins showed good correlation within the experimental error range of determined Mr and pI values with those predicted from their amino acid sequences but some showed significantly reduced experimental mass, suggesting that they were products of proteolytic degradation. For example, 78-kDa glucose-regulated protein (BIP), vimentin, and actin were represented by both full-length proteins and fragments whereas calmodulin and heat shock cognate 71-kDa protein were represented only by fragments (Table). Because cellular proteins were always extracted in the presence of protease inhibitors and protein fragments were detected only in ouabain-treated VSMCs, it is likely that proteolysis took place in living cells, but at this point we have no explanation for this phenomenon. Also, a group of histones showed remarkable difference between the theoretical (10.5 to 11.7) and experimentally determined (4.5 to 5.6) pI values. Posttranslational modifications such as acetylation and methylation of the basic amino acid residues frequently found in the histone sequences could be partially responsible for this discrepancy.30 It is also possible that during a separation in the first dimension histones stay as protein-DNA complexes with acidic pI values. After denaturing with SDS before the second dimension, histones lose their ability to bind DNA and run on the SDS PAGE according to their molecular weight. Experimental pI values in the acidic pH range were also determined for histones by other authors.31 No changes in posttranslational modification were detected within the amino acid sequence observed by MS, but this does not preclude changes in the other regions of the protein.
We observed that ouabain changed the expression level of different groups of cellular proteins including cytoskeleton and associated proteins (vimentin, actin, vinculin, and myosin); plasma membrane proteins and receptors (annexin I, α-macroglobulin receptor-associated protein); DNA-binding proteins and proteins involved in the regulation of DNA synthesis (histones, nucleophosmin, and prohibitin); Ca2+-binding proteins (calmodulin), as well as glycolytic and mitochondrial enzymes (GAPDH, α-enolase, and aldehyde dehydrogenase). One spot was identified as a rat analogue of a hypothetical human protein FLJ14844. However, a group of heat shock proteins (GRP78, HSC70, and HSP47) and, in particular, mitochondrial stress-70 protein, mortalin, also called GRP75 and PBP-74 (reviewed in Wadhwa et al32), were most significantly induced by ouabain. This assignment was based on online nano–LC-MS-MS results. An example of this is shown in Figure 2 for 2 of the 13 MS-MS spectra obtained from spot 2 (Table). In both cases, the MS-MS spectra are dominated by cleavage of the peptide bond giving rise to y-type fragment ions with charge retention on the C-terminus segment of the precursor ion. Because previous works showed that mortalin overexpression could rescue NIH 3T3 and carcinoma cells from apoptosis induced by serum starvation, UV irradiation, and γ-irradiation,33,34⇓ we have concentrated our further studies on this particular protein.
Expression of Mortalin in Ouabain-Treated VSMCs
Northern blot showed a 5-fold increase of the mortalin RNA after 2 hours of ouabain treatment (Figure 3A), whereas Western blot demonstrated that the level of mortalin protein started to increase already after 30 minutes of treatment and reached its maximum 3 hours after (Figure 3B).
Anti-mortalin immunofluorescence in VSMCs was associated with punctate structures colocalized with the mitochondrial marker MitoTracker Red (Figure 3C) and distributed with a gradient of concentration from the plasmatic to the nuclear membrane. The cells treated with ouabain for 2 hours showed similar localization of mortalin, but the intensity of staining significantly increased, reflecting a high expression level of the protein (Figure 3C).
Expression of Mortalin in VSMCs Results in Their Increased Resistance to Serum Deprivation–Induced Apoptosis
We expressed mortalin in VSMCs to understand whether this protein is involved in the inhibition of apoptosis. Preliminary experiments showed that VSMCs are highly resistant to transfection. Under optimal conditions, only 25% to 30% of cells expressed a reporter β-galactosidase gene (not shown). To overcome this problem, we used VSMC-E1A cells, stably transfected with E1A-adenoviral protein,28 which showed 96% to 98% transfection efficiency under similar conditions (not shown). Another advantage of using VSMC-E1A cells was their high susceptibility to apoptosis induced by serum deprivation compared with wild-type VSMCs.28 VSMC-E1A cells showed a similar effect to wild-type VSMCs’ effect of ouabain on the development of apoptosis.20
VSMC-E1A cells were transfected with pCMV-SPORT-Mot vector containing a full-length human mortalin cDNA. Western blot analysis of the transfected cells (Figure 4) revealed expression of a full-size mortalin protein. Forty-eight hours after transfection, cells were placed in serum-free medium and at time intervals indicated on Figure 5 were fixed and stained with DAPI. The number of apoptotic cells was then counted either based on chromatin condensation using epifluorescence microscopy or on cell morphology using phase-contrast microscopy (Figures 5A and 5B). Both methods showed that the rate of apoptosis in VSMC-E1A cells transfected with mortalin cDNA was significantly reduced during the first 6 hours after serum deprivation. For example, only ≈15% of mortalin-transfected cells showed condensed nuclei 2 hours after serum deprivation compared with >30% in mock-transfected cells (Figure 5A). During the first 4 hours, the effect of mortalin on cell survival was comparable to that of ouabain, but ouabain provided long-term (up to 24 hours) protection (Figure 5B). As in the case of ouabain treatment, transfection of VSMC-E1A cells with mortalin cDNA resulted in almost 2-fold inhibition of caspase-3 activity measured 2 hours after serum deprivation (Figure 5C).
Because the commercially available monoclonal anti-human mortalin antibodies were also specific against the endogenous rat protein, it was difficult to discriminate between transfected and nontransfected cells using immunofluorescence microscopy. Therefore, to estimate the rate of apoptosis specifically in mortalin-expressing cells, we transfected VSMC-E1A cells with mortalin cDNA using a pIRES2-EGFP-Mot vector that allowed mortalin and a transfection marker, green fluorescent protein (GFP) to be translated from a single bicistronic mRNA. As in the previous experiment, 48 hours after transfection cells were incubated in serum-free medium, fixed, and stained with DAPI. Immunofluorescence microscopy showed that GFP-positive cells had much higher resistance to apoptosis induced by serum deprivation than GFP-negative nontransfected cells (not shown).
Mortalin Inhibits Apoptosis in VSMCs via the Inactivation of p53
Previous studies have demonstrated that serum deprivation–induced apoptosis in VSMCs, in particular in VSMC-E1A cells, is executed by a p53-dependent mechanism.28,35,36⇓⇓ In agreement with this, we found that serum deprivation of wild-type or mock-transfected VSMC-E1A cells resulted in rapid induction of p53 (≈4-fold increase 1 hour after deprivation; Figure 6A). In contrast, the induction of p53 in mortalin-transfected cells was delayed for about 6 hours (Figure 6A), suggesting that mortalin may inhibit apoptosis in VSMCs by reducing the level of p53. Immunofluorescent microscopy showed the predominantly nuclear localization of p53 in serum-deprived VSMCs, but the majority of mortalin-transfected cells showed a juxtanuclear staining pattern (Figure 6B). Colocalization of p53 and mortalin previously observed in mortalin-transfected NIH 3T3 cells32,37⇓ suggested the cytoplasmic retention/abrogation of the nuclear translocation of p53 whereas recent studies directly showed that the N-terminal region of mortalin binds to a C-terminal cytoplasmic sequestration domain of p53.38
A number of VSMCs in vessel walls are regulated by the balance between proliferation, senescence, and apoptosis. These cells have an effective mechanism to discriminate between these conditions in response to diverse stimuli such as mechanical forces, reactive oxygen and nitrogen species, cytokines, and growth factors. The tumor suppressor p53 controls at least in part both growth and senescence of VSMCs39–43⇓⇓⇓⇓ as well as their apoptosis.35,44–47⇓⇓⇓⇓ In this work, we demonstrate the existence of a new [Na+]i/[K+]i-responsive signaling pathway for the control of apoptosis in VSMCs that allows these cells to inactivate p53 through the induction of HSP70 family protein, mortalin.
To characterize changes in a proteome of VSMCs induced by ouabain treatment that inversed [Na+]i/[K+]i ratio and inhibited serum deprivation–induced apoptosis, we analyzed subsets of hydrophilic and hydrophobic VSMC proteins from control and ouabain-treated cells by 2DE. The 2DE-MS approach permits studying only a part of the cellular proteome, because proteins of medium to low abundance as well as transmembrane proteins are usually not detected.48 Other methods such as the recently described isotope-coded affinity tag (ICAT) approach49 should be implemented to complete the characterization of the VSMC proteome. Nevertheless, the 2DE-MS method revealed that ouabain treatment results in significant changes in the expression level of numerous VSMC proteins. One of those proteins, mortalin, was directly implicated in apoptosis regulation.
Northern and Western blotting and immunocytochemistry all have confirmed that the inhibition of VSMC apoptosis after sustained increase of [Na+]i/[K+]i ratio by ouabain is accompanied by potent induction of mortalin expression. Previous studies have shown two different types of intracellular localization of mortalin, pancytosolic and perinuclear. In mortal cells, the protein is widely distributed in the cytoplasm whereas in immortal cells the protein shows mitochondrial juxtanuclear distribution, or fibrous staining with perinuclear concentration.50 Perturbation of the pancytosolic and overexpression of the perinuclear form were observed in most of the tumors and tumor-derived cell lines, showing progressive increase with malignancy.33,37,51⇓⇓
In our studies, mortalin-transfected VSMCs showed a 6-hour delay in the development of apoptosis after serum deprivation. Within this period, the amount of morphologically changed apoptotic cells, or cells that exhibited chromatin condensation and blebbing of the nuclei, was reduced about 2-fold compared with control cells. Caspase-3 activity level was also reduced showing that the development of apoptosis was suppressed at a step before the activation of this enzyme.
Western blotting and immunocytochemistry revealed that the expression of p53 in mortalin-transfected cells was delayed and the expressed protein was not located at the nuclei but rather showed perinuclear distribution suggesting binding of mortalin to p53, which results in unfolding and inactivation of the latter protein.52 The inactivation of p53 by mortalin in VSMCs is apparently a part of their physiological response to inversion of the [Na+]i/[K+]i ratio and may be also a component of their defensive mechanism against apoptosis. The changes in p53 expression have not been directly detected in proteomic studies probably because the expression level of p53 was below the detection limit of the 2DE-MS method.
The protective effect of mortalin was lower and less prolonged than that of ouabain, suggesting that mortalin induction is only one of the antiapoptotic mechanisms triggered by elevation of [Na+]i/[K+]i ratio in VSMCs. Some of these mechanisms could involve chaperones of HSP70 family such as HSC70 and GRP78 (BIP), which directly bind p53 in the process of the antiapoptotic response (reviewed in Zylicz et al53). Both HSC70 and GRP78 (BIP) were found induced in ouabain-treated VSMCs in this study (Table and Figure 1). Recent data also showed that co-chaperone HSP40 affects interactions of HSP70 and HSP90 with the wild-type p53.54 In particular, HSP90 and HSP40 displace each other from the complex with p53, suggesting that they may compete for the same p53 domains.54 Interestingly, the expression level of rat HSP40 protein, GP46 was 2-fold reduced by ouabain treatment (Table and Figure 1), which could additionally facilitate the formation of mortalin-p53 complexes. Another potential antiapoptotic protein induced by ouabain is a mitochondrial chaperone, prohibitin (PHB). Originally identified as putative negative regulator of the cell cycle, PHB was recently shown to protect B cells against apoptosis caused by tropoisomerase inhibition.55 Experiments that should demonstrate whether the induction of PHB, HSC70, and BIP and suppression of GP46 are also part of ouabain-induced antiapoptotic mechanism are presently in progress in our laboratory.
It is tempting to speculate that mortalin-p53 interactions may be involved in the regulation of apoptosis, senescence, and proliferation of VSMCs in several different types of remodeling. For example, both early senescence and p53-induced apoptosis observed in the VSMCs from atherosclerotic plaques39 are consistent with the “senescent phenotype” of cells that have predominantly pancytoplasmic distribution of mortalin. In contrast, reduced apoptosis and increased proliferation of VSMCs during neointimal response to injury could be due to the p53 inactivation by the perinuclear mortalin pool. If this is the case, mortalin may be a potentially important pharmaceutical target for selective modulation or inhibition of apoptosis in the vessel wall. In particular, inhibitors of mortalin may induce apoptosis in VSMCs and therefore be helpful for limiting vascular lesions associated with restenosis.
This work was supported in part by an operating grant from the Canadian Institutes of Health Research MT-38107 to A.V.P. and MT-10803 to P.H. and S.N.O. as well as by the equipment grant from the Canadian Foundation for Innovation to A.V.P. V.S. acknowledges a postdoctoral fellowship from the Fonds de la Recherche en Santé du Québec. The authors thank Dr Mila Ashmarina for stimulating discussions and Liliane Gallant for help in preparation of the manuscript.
Original received April 17, 2002; resubmission received September 4, 2002; revised resubmission received October 4, 2002; accepted October 10, 2002.
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