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VASCULAR BIOLOGY
-mediated apoptosis in vascular smooth muscle cells requires p73
1Division of Nephrology, Department of Internal Medicine, 2Immunology Graduate Group, and 4Cancer Center, University of California, Davis; 3Department of Pathology, University of California, San Francisco; and 5Department of Veterans Affairs Northern California Health Care System, Sacramento, California
Submitted 28 September 2004 ; accepted in final form 10 February 2005
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ABSTRACT |
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, which is known to contribute to apoptosis in vascular smooth muscle cells, has been shown to be intimately involved in the atherosclerotic process, being present at elevated levels in human atheroma as well as possibly being responsible for plaque rupture, a clinically devastating event. In light of our earlier finding that p73 is a proapoptotic protein in vascular smooth muscle cells, which are involved in plaque progression as well as rupture, we asked whether TNF- mediates apoptosis in these cells through p73. We now show that p73 is present in spindle-shaped cells within human atheroma, and p73
, an isoform that is pivotal in both apoptosis and growth suppression, is induced in vascular smooth muscle cells in vitro by serum but not by PDGF-BB. In addition, TNF-, when added to these cells in the presence of serum-containing media, increases p73 expression and causes apoptosis in both rat and human vascular smooth muscle cells. Inhibition of p73 activity with a dominant inhibitory NH2-terminally deleted p73 plasmid results in markedly decreased TNF--induced apoptosis. Thus p73 is likely a mediator of the apoptotic effect of TNF- in the vasculature, such that future targeting of the p73 isoforms may ultimately prove useful in novel atherosclerosis therapies. atherosclerosis; inflammation; plaque
Because apoptosis is a major contributor to the process of plaque rupture, research into the mechanism and mediators of this process in vascular smooth muscle (VSM) cells is an area of pivotal importance in atherosclerosis research. TNF- is a 17-kDa proinflammatory cytokine that acts as a homotrimer on two distinct membrane bound receptors, TNF-R1 and TNF-R2 (4). This cytokine is detected at elevated levels in atherosclerotic vessels in rabbits (19), as well as in human atheroma (1, 30), and it may contribute to rupture of these plaques. In vitro studies have shown that TNF- stimulates VSM cell migration (14) and, perhaps more importantly to its role in atherosclerosis, that intimal smooth muscle cells exposed to TNF- undergo apoptosis at a higher rate than unexposed medial smooth muscle cells (25).
p73 is a family of proteins that are closely related in sequence, as well as in some functions, to the tumor suppressor p53. As such, p73 activates many p53 target genes and, similarly to p53, causes growth suppression and apoptosis in a variety of cell lines (13, 15) including VSM cells (6). Our earlier findings that p73 is present in human atherosclerotic plaque lysate (40) and that overexpression of p73 causes apoptosis in rat thoracic aorta smooth muscle cells (6) led us to investigate the role of p73 in VSM cell apoptosis.
At present, six alternatively spliced p73 mRNAs have been identified in normal cells, named , , 
, 
, 
, and 
, but the function of the various isoforms in VSM cells is just beginning to be investigated. We have shown (6) that overexpression of p73 in VSM cells results in apoptosis, but the -isoform of p73 may be more important than other isoforms in apoptosis (28) and growth suppression (9, 16). Indeed, in several assays p73 has been shown to have stronger transcriptional activity than p73, possibly because the longer COOH-terminal region of full-length p73 is inhibitory (7). For this reason, we examined primarily this isoform in the current study. We now show that, on serum stimulation, both human and rat VSM cells express p73. In addition, treatment of VSM cells with TNF- in the presence of serum-containing media further upregulates p73 levels in these cells, and p73 is in fact required for the full effect of TNF- on apoptosis. In light of the data presented in this study, p73 can be considered a target for further research into the apoptotic pathways, and thus fibrous cap stability, of atherosclerotic lesions.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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was obtained from R&D Systems (Minneapolis, MN), and human recombinant TNF- was obtained from Roche Applied Science (Indianapolis, IN). Mouse monoclonal p73 (Ab-1), which recognizes p73 and p73-p73 complexes, and p73 (Ab-3) and rabbit anti-human p73 (Ab-4), which recognizes both p73 and p73 antibodies, and GeneJuice transfection reagent were purchased from Oncogene Research (Boston, MA). Mouse monoclonal anti-human recombinant full-length p21Waf1/Cip1 antibody was obtained from Upstate Biotechnology, and rabbit anti-human polyclonal p57kip2 antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal poly(ADP-ribose) polymerase (PARP) antibody was obtained from Cell Signaling Technology (Beverly, MA). The CaspACE colorimetric assay system was obtained from Promega (Madison, WI). All other reagents were obtained from Sigma (St. Louis, MO). Cell culture. Rat VSM cells, passages 19–30, were cultured in growth medium containing DMEM, 10% FBS, and 50 U/ml penicillin-streptomycin. Human VSM cells, passages 5–8, were grown in Clonetics proprietary growth medium. To serum starve the cells, they were placed in serum-free medium containing DMEM, 20 mM HEPES (pH 7.4), 5 mg/ml transferrin, 0.5 mg/ml BSA, and 50 U/ml penicillin-streptomycin for 48 h. Serum-starved cells were stimulated with either growth medium or 40 ng/ml PDGF-BB for the times indicated.
Western blotting.
A10 cells were grown to 60–70% confluence. The cells were then treated with 10 ng/ml rat or human TNF- for the times indicated. Media and washes were collected and centrifuged to collect the dead cells. Cells were lysed, and then equal protein amounts were loaded and electrophoresed and immunoblotted with the Bio-Rad DC protein assay (Hercules, CA) as previously described (41). The membranes were blocked in 5–10% nonfat dry milk for 1 h at room temperature and probed with appropriate antibodies in 5% nonfat dry milk overnight at 4°C. The membranes were then probed with horseradish peroxidase-tagged anti-mouse or anti-rabbit IgG antibodies (Bio-Rad) diluted 1:15,000 in 2.5–5% nonfat dry milk for 1 h at room temperature. Chemiluminescence was detected by enhanced chemiluminescence (Amersham Biosciences).
Transfections.
VSM cells were seeded into 35-mm dishes at 50–70% confluence 1 day before transfection. Transient transfection using Gene Juice transfection reagent with no plasmid, pcDNA-green fluorescent protein, pcDNA hemagglutinin (HA)-tagged p73 (7), and pcDNA HA-tagged 
Np73 (24) was performed according to the manufacturer's protocol. Fresh medium was added after 6-h incubation with the transfection complex. Cells were lysed 24–48 h after transfection.
Caspase activity.
A10 cells were grown in 100-mm dishes until 
50–70% confluence. Rat TNF- (10 ng/ml) was added for the times indicated. The media and washes were collected and centrifuged to collect the dead cells. Both cells and media, as well as washes (containing dead cells), were combined and then lysed. Caspase activity was detected colorimetrically on equal quantities of lysate protein with the CaspACE assay system according to the manufacturer's protocol.
Immunohistochemistry. Internal Review Board approval was obtained from the University of California, Davis, and the University of California, San Francisco, and we searched the pathological records from the University of California, San Francisco, from 1995 to 2002 and selected representative sections from 20 different carotid endarterectomy specimens. Of these, 14 of 20 clearly demonstrated characteristic features of arterial intimal plaque on hematoxylin and eosin-stained sections. Routine histological staining and immunohistochemistry were performed on 5-µm-thick tissue sections cut from formalin-fixed paraffin-embedded tissue blocks and placed on Superfrost/Plus slides.
The immunohistochemical staining was performed using the avidin-biotin peroxidase method. -Smooth muscle actin (SMA; Dako, Carpinteria, CA) was used to evaluate for the presence of cells of muscular derivation, and a polyclonal antibody recognizing both p73 and p73 (Ab-4) was used to identify the presence of p73 in these same cells. After deparaffinization and blocking of endogenous peroxidases, tissue sections were steam treated and allowed to stand in buffer for 1 additional hour. p73 antibody diluted 1:3,500 and SMA antibody diluted 1:100 were incubated with the tissue sections at 4°C overnight. Biotinylated anti-mouse and avidin-biotin complex (Vector, Burlingame, CA) were applied to each section and developed with the peroxidase reaction, using diaminobenzidine as chromogen according to standard methods. Sections were counterstained with hematoxylin. An internal positive control for SMA included small, intact arteries present in the plaque material. Elastica-van Gieson (EVG) staining was used to identify elastic fibers normally present in the intima and media of vessels.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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is increased in VSM cells after serum stimulation.
We showed previously (40) that on serum but not PDGF-BB stimulation of VSM cells, p73 is increased in a time-dependent manner, but the antibody used in our original study likely recognized both - and -isoforms of p73 and thus did not differentiate between the two. Because p73 has been shown to be important in apoptotic (28) as well as growth-suppressive (9, 16) pathways, we thus asked whether p73 was increased under these conditions. Using antibodies specific to p73 or p73, we performed immunoblotting of serum-starved rat VSM cells that had been stimulated at various times with 10% serum. Because no bands were initially present in the p73 blot, we utilized a tetracycline-overexpressed (tet-off) p73 protein (6) as a positive control. Consistent with our previous study in these cells (40), serum-stimulated VSM cells showed increased levels of p73 at 1–2 h after stimulation (Fig. 1A); however, p73 was not present under similar stimulatory conditions (Fig. 1B). Furthermore, as in the original study, there was no increase in p73 when the cells were stimulated with PDGF-BB and no increase in serum-stimulated p73 induction with the addition of PDGF-BB (Fig. 1C). The most likely explanation of this disparity is that there is a protein or other molecule present in serum that increases p73 and that is not PDGF-BB. As examined in this study, it is possible that an inflammatory mediator, such as TNF-, accounts for this difference.
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and p73. Hematoxylin and eosin staining of a sample is shown in Fig. 2A. The distribution and intensity of staining of p73, -SMA, and EVG were evaluated in these samples. p73 immunoperoxidase staining was of greatest interest among these stains as its presence was uniformly distinct in fusiform or spindle cells, with focal areas of more intense staining in some of the larger fusiform cells denoted by arrows (Fig. 2, B and C). SMA showed diffusely positive staining in these same cells, supporting the hypothesis that they are most likely of smooth muscle derivation; in addition, SMA was strongly positive in the walls of adjacent small muscular vessels in the endarterectomy specimens, as would be expected (data not shown). The EVG stain enhanced the architectural pattern of the intimal plaque and confirmed the presence of delicate elastic fibers unevenly distributed in the disrupted neointimal regions involving plaque material and interspersed between the spindle cells (data not shown). These findings demonstrate the presence of p73 in cells that are likely of smooth muscle origin found within carotid atherosclerotic plaque. Thus p73 is likely playing a role in human atherosclerotic plaque formation and/or maintenance through its expression in VSM cells contained within the plaque.
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added in presence of serum increases p73 in continuously growing VSM cells.
In light of our previous finding that overexpression of p73 in VSM cells induces apoptosis (6), we next looked at the possibility that TNF- may involve p73 in its VSM cell apoptotic signaling pathway, as has been reported in fibroblasts and colorectal carcinoma cells (3). To ascertain whether p73 is increased in response to TNF-, we incubated non-serum-starved VSM cells (to replicate the in vivo milieu) with complete medium alone or with TNF- alone for 2–5 h and immunoblotted the cell lysate with the antibody specific for p73. The time zero points had no TNF- or fresh serum added and the cells were immediately lysed, whereas the later time points had TNF- alone or fresh serum added for the indicated time followed by lysis. There was no change in p73 when the cells were incubated under these conditions (Fig. 3A), consistent with other reports showing that TNF- alone does not cause apoptosis (11, 17, 26).
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when added alone to VSM cells (11, 17, 26), we next asked whether the addition of TNF- in the presence of fresh serum-containing medium causes alterations in p73 levels. p73 levels in VSM cells treated with TNF- in the presence of fresh serum for the indicated times were markedly increased, starting 2 h after incubation and persisting for 5 h, compared with untreated cells (t = 0) (Fig. 3B). A homologous result was seen in human VSM cells with human TNF- (Fig. 3B). Thus TNF- added to non-serum-starved VSM cells in the presence of fresh serum, mimicking the in vivo milieu that occurs during inflammatory conditions, increases p73.
TNF- causes apoptosis in VSM cells.
TNF- has been shown to induce apoptosis in intimal VSM cells (25), which can lead to fibrous cap instability and plaque rupture as discussed above. To determine whether such an effect is mediated by p73, we incubated proliferating VSM cells (replicating the in vivo milieu) with TNF- plus serum-containing medium for 24–96 h and examined the lysate for PARP cleavage, an early event in the apoptosis cascade, by immunoblotting. Compared with serum-only stimulated cells, TNF--treated cells treated in the presence of serum showed an increased cleavage of the 113-kDa PARP band, an early event in apoptosis, with the maximal difference in PARP cleavage between TNF- and serum-alone stimulation being at 96 h (Fig. 4A). There was some apoptosis seen in serum-only stimulated cells, probably due to the length of time in the absence of fresh serum, but in all cases PARP cleavage in cells incubated with serum + TNF- was greater than with serum alone. To confirm this finding with another measure of apoptosis, we also examined caspase-3 activation in the same cell lysate. As expected, caspase-3 activity paralleled PARP cleavage in cells treated with TNF- (Fig. 4B). Thus VSM cells undergo apoptosis on treatment with TNF-.
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-induced apoptosis is dependent on p73.
In light of data in mouse thymocytes that TNF- activates the proapoptotic function of p73 (3), we next asked whether the observed proapoptotic effect of TNF- in VSM cells (Fig. 4) requires functional p73. In these experiments, we used an NH2-terminally truncated Np73 expression plasmid. This isoform has been shown to act in a dominant inhibitory fashion against full-length p73 by direct competition for DNA binding of the nontruncated forms (36), such that the NH2-terminally truncated form inhibits apoptosis (24, 27). In addition, it has been shown that Np73 causes stabilization of both p73 and p73 proteins (31). Because the transactivation inhibitory function of Np73 toward p73 depends on the formation of stable protein-protein complexes (24, 36), it is likely that Np73 is inhibitory toward p73 and p73. Indeed, Np73 is more ideal in this situation because it has a much stronger stabilizing effect toward p73 isoforms than Np73 (31). Furthermore, Np73 (unlike Np73) has an independent effect on growth suppression and is itself capable of transactivation (21), so that its use in this context would yield ambiguous results.
VSM cells were transfected with the Np73 plasmid before treatment with TNF- in serum-containing medium. Transfection efficiency was determined with an antibody recognizing the COOH terminus of p73 and thus also the NH2-terminally truncated forms. Although TNF- caused significant apoptosis in mock-transfected VSM cells, transfection of the cells with the dominant-negative Np73 plasmid showed a DNA concentration-dependent decrease in apoptosis in response to TNF- (Fig. 5A). Successful transfection of the Np73 plasmid was confirmed by immunoblotting (Fig. 5B).
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and a subsequent decrement in its level after transfection of the dominant-negative p73 construct with 1.5 µg of Np73 (Fig. 5C). To confirm that the Np73 plasmid that we used, which has no epitope tag, was actually being translated after transient transfection, we used a cell line that does not express significant endogenous p73 such that there would be no interference between endogenous p73 and the transfected human forms. This Met-1 mouse mammary tumor line (42) was transiently transfected with p73, Np73, or both plasmids and Western blotted with a p73 antibody that recognizes both isoforms. There was a transfection dose-dependent increase in Np73 that migrated at 65 kDa in cells transfected with the Np73 plasmid (Fig. 6A). Cells transfected with the p73 plasmid showed p73 protein that migrated at a higher molecular weight, and in cells transfected with both Np73 and p73, it can be clearly seen that both are expressed (Fig. 6B). Thus the Np73 plasmid used in this experiment is expressing the desired protein.
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Np73 isoform probably acts in a dominant-negative fashion toward all nontruncated forms, we cannot differentiate whether it is p73 or p73 (or both) that is being inhibited in this experiment (see discussion above). However, because there were very low (if any) levels of p73 in these cells under serum-stimulated conditions (Fig. 1), we can conclude that TNF-, which causes apoptosis in VSM cells, does so at least in part through its activation of nontruncated forms of p73, most likely p73. |
DISCUSSION |
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Whereas before our earlier findings p73 had not been described to be present in vascular tissues, p53 induction has been associated with apoptotic changes in arterial aneurysms (reviewed in Ref. 23). p53, which is closely related in structure (but not in all functions) to p73, is also markedly increased after balloon angioplasty of the rabbit iliac artery in a manner that parallels apoptosis (32). Because p73 expression is often observed under similar situations when p53 is present, it is likely that p73 protein also plays a role in VSM cell growth and in the pathogenesis of atherosclerosis.
Although p73 and p53 have different ultimate effects, there is evidence that p73 can activate many, although not all, p53 target genes, including p21 (35). p73, in particular, has been shown to activate expression of the cyclin kinase inhibitor p57Kip2 in cancer cell lines, whereas p53 does not (2); however, the function of p57Kip2 in nontransformed and vascular cell lines, both stimulated by p73 and not, is not known. There are also situations in which p73 and p53 induce expression of the same proteins, such as p21waf1/cip1 (43), consistent with our data showing that dominant-negative p73 at high transfection levels attenuates the TNF- (and thus presumably p73)-dependent increase in p21 (Fig. 5C). The p21 story is made more complicated by our work showing that this cyclin kinase inhibitor can act in both a pro- and an antiapoptotic manner (reviewed in Ref. 39), such that p21's function downstream of p73 is not obvious. Whether the differences in p73 function and tissue specificity are wholly dictated by these target genes remains to be established.
TNF- is known to cause apoptosis in VSM cells, and this has been postulated to be a contributor to plaque rupture (1). It has also been hypothesized that apoptosis caused by activation of endogenous TNF receptors in VSM cells (25) may be a mechanism by which injured arteries limit accumulation of these cells. Our finding of the requirement for TNF- to be added to VSM cells in conjunction with serum for p73 to be increased (and presumably for apoptosis to occur) is consistent with other reports showing minimal effect alone but a synergistic effect of TNF- when added to VSM cells 1) overexpressing truncated I
B (26) and 2) in combination with a proteosome inhibitor (17) or interferon- (11).
The p53 class of tumor suppressor proteins are responsible for mediating the cellular response to DNA damage, through the cyclin kinase inhibitors as well as other downstream proteins (reviewed in Ref. 39). Although p73 is dissimilar to p53 in a variety of its effects, the two cousins both have the ability to cause apoptosis under certain conditions. The mechanisms of the proapoptotic effects of p53 and p73 may be different, especially in light of the complex pattern of both pro- and antiapoptotic isoforms generated by differential splicing and the presence of alternative promoters on the p73 gene (reviewed in Ref. 34).
Although there is abundant information concerning the mechanism and tissue specificity of apoptosis by p73, data on the apoptotic function of p73 in the human vasculature are sparse. Using a conditional expression system, we showed previously (6) that overexpression of p73 leads to apoptosis in rat VSM cells, yet others have shown that apoptosis induced by E2F1 is independent of p73 in human VSM cells (33). p73 may also be important in tumor progression via its effect on tumor vasculature, as evidenced by the finding that overexpression of this protein increases VEGF (38), a result consistent with the work of other investigators showing a close association between VEGF and p73 expression in colorectal carcinoma (12). Because both VEGF and TNF- are vasoactive and inflammatory mediators, whether a proapoptotic effect of TNF- through p73, as shown by our data, is important in countering tumor angiogenesis remains to be seen.
Our previous reported findings of increased p73 in VSM cells exposed to serum may have been due to the recognition of both p73 and p73 by the antibody in the cells used at earlier passage (40) or by the lack of specificity of the antibody used in the earlier studies. Now that we have a positive control for p73 (6), we are able to determine that p73 is the predominant species in this cell line in response to a mitogenic stimulus (Fig. 1). On the basis of the known proapoptotic roles of both p73 and p73, the potential function of p73 in VSM cell growth likely relates to growth suppression and/or apoptosis. As mentioned above, the ability of p73 to affect both cyclin kinase inhibitors p21, as we confirm here, and p57 (in cancer cells; Ref. 2) may actually result in either cell cycle arrest or apoptosis similar to p53. However, the lack of tumor formation in p73-knockout models and in primary human tumor data argues against p73 functioning as a classic tumor suppressor (34). Until further data are obtained showing the effect of overexpressed p73 in VSM cells, as we have done for p73 (6), this remains an open question in this cell type.
Our finding that the apoptotic function of a pluripotent mediator of inflammation, TNF-, is mediated by p73 is consistent with the known function of p73 in growth suppression and apoptosis. Our data are also consistent with a recent study in a human B cell lymphoblastoid cell line (Ramos cells) in which TNF- also increased p73 levels (5), suggesting that this phenomenon may be more generally applicable. Although we were unable to completely attenuate the TNF--induced apoptotic effect with a dominant-negative plasmid approach, our work indicates that the effect of TNF- specific to the vasculature requires functional p73. Thus artificial regulation of p73 may be a viable approach to improving the stability of a plaque or fibrous cap that may occur after angioplasty, for example. This is especially important, given the high rate of angioplasty failure, which in some cases may be due to plaque rupture.
Consistent with our findings, activation of TNF- receptors has been shown to increase the rate of intimal VSM cell apoptosis (25). In that study, TNF- mRNA, but not VSM cell growth rate, was increased in response to other inflammatory mediators, interferon- and interleukin-1. However, VSM cells that have been induced to proliferate by balloon injury express TNF- (37). Other investigators have shown that treatment of VSM cells with TNF- alone did not result in apoptosis (11), a disparity that may be due to cell type, passage number, or other conditions. Although our data are consistent with other studies and support a definite role of TNF- in VSM cell apoptosis, the nature of the effect of this cytokine on cell proliferation, also important for atherosclerotic lesion pathogenesis, is not known and was not addressed in this study.
The location of p73 within the nuclei of musclelike cells within plaque tissue, as described in our study, suggests that this protein may be playing a growth- or apoptosis-modulatory function in this tissue. Should p73 be directing these cells into an apoptotic pathway, ultimate targeting of this protein for attenuation by techniques such as antisense or small interfering RNA may lead to a salutary effect by decreasing plaque rupture. On the other hand, p73 may be mediating an antiproliferative effect on the VSM cells, which in the case of plaque growth may indeed be a desired outcome. In either case, further research into the role of p73 in the atherosclerotic process is likely to yield data that may be used in future therapy of this devastating disease.
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ACKNOWLEDGMENTS |
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Np73 plasmid, Dr. G. Melino for supplying the p73 plasmid, Dr. B. Vogelstein for the tet-off p73 plasmid, and Christabel Moy and Ben Davis for technical help and critical reading of the manuscript. |
FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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