1 Division of Nephrology, Department of Internal Medicine, 2 Cell and Developmental Biology Graduate Group, and 3 Cancer Center, University of California, Davis 95616; and 4 Department of Veterans Affairs, Northern California Health Care System, Mather, California 95655
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ABSTRACT |
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Abnormal vascular smooth muscle (VSM) cell proliferation contributes to the development of atherosclerosis and its associated disorders, including angioplasty restenosis. The tumor-suppressor protein p53 has been linked to the development of atherosclerotic lesions, and its homolog, p73, is proving to have contrasting functions in a variety of tissues. As an outgrowth of our previous finding that p73 is increased in serum-stimulated VSM cells and human atherosclerotic tissue, we examined p73 overexpression in VSM cells to elucidate causality of p73 expression with growth response. Overexpression of p73 results in decreased cell cycle transit and is accompanied by apoptosis. The apoptotic changes in p73 overexpressing VSM cells are independent of p53 and are associated with a decrease in levels of p21waf1/cip1. In conjunction with our previous data finding that p73 is increased in serum-stimulated VSM cells, this work suggests a role for p73 in vascular proliferative diseases.
p21; p53; atherosclerosis; tet-off; proliferation
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INTRODUCTION |
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SINCE ITS CLONING IN 1979 (14, 16), the p53 gene and its associated tumor-suppressor protein have been extensively studied. The critical importance of p53 over evolutionary time is evident by its presence in more than 23 species ranging from mollusks to humans (26). But whereas p53 has been known for many years to function as an arrestor of cell cycle transit or a promoter of apoptosis, its newly described homologs, p73 and p63 (12), have been shown in some cases to have effects quite opposite to p53.
p73 has been studied extensively in cancer cells, where it has been shown under different conditions and with cell lines to be either pro- or anti-apoptotic (9, 11, 17, 21, 39), but the function of this protein in "normal" cell lines has not been adequately investigated. Given its homology to the tumor-suppressor protein 53, the finding that p73 is frequently overexpressed in human tumors (4, 29) suggests that this protein plays a growth regulatory role at least in this tissue, yet whether it contributes positively to cell growth or suppresses proliferation is not known. The issue is further complicated by the finding that the p73 protein, represented by its dual promoter and alternatively spliced forms, seems to have different, sometimes opposite, functions in different cell types. Thus it has been difficult to reconcile the similarities of these tumor proteins on downstream proteins with the findings, in particular, that p53 knockout mice develop spontaneous tumors at high frequencies in multiple tumors (10), whereas p73 knockouts show no increased susceptibility to spontaneous tumorigenesis (37).
p53 has been shown to be present in the vasculature (1) and exerts a growth-suppressive effect in human aortic vascular smooth muscle (VSM) cells (19). p53 is also markedly increased after balloon angioplasty of the rabbit iliac artery in a manner that parallels apoptosis (25). Thus this protein has been described as a possible "functional link" between VSM cell growth and apoptosis (19), as well as an important protein in the development of coronary restenosis (27). With the exception of our earlier work, in which we demonstrated that p73 is increased in serum-stimulated VSM cells (32), p73 has not been described otherwise in the vasculature and has been shown to be expressed only at very low levels in 51 other normal tissues (13). Although our previous work suggests that p73 is involved in some manner in vascular growth control, a causal relationship between p73 expression and VSM cell growth has not been established. The nature of such a relationship is not obvious, due to the observed cell type differences in the function of p73, but it is likely that p73 is either causing VSM cell growth stimulation or is a homeostatic response in an attempt to attenuate this response.
Using a conditional overexpression model, we now show that p73 triggers a p73 expression-dependent inhibition of VSM cell S-phase entry (as a measure of cell cycle transit) and induction of apoptosis, independently of p53 but associated with a decrease in the level of the cyclin-dependent kinase inhibitor (and newly characterized "survival" protein) p21waf1/cip. The fact that p73 is both induced in serum-stimulated VSM cells and is apoptosis promoting suggests that this response is a means by which vascular (and perhaps other) cells attenuate overexuberant VSM cell growth.
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MATERIALS AND METHODS |
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Materials.
Tet-off expression plasmids pBI-p73wt/EGFP (expressing wild-type p73)
and tTA-IRES-Neo were obtained as kind gifts from Dr. Bert Vogelstein
(Baltimore, MD). pTK-hygro was obtained from Clontech (Palo Alto, CA).
Mouse monoclonal p21 antibody and human recombinant PDGF-BB were
obtained from Upstate Biotechnology (Lake Placid, NY). Mouse monoclonal
p53 and rabbit polyclonal caspase-3 antibodies were from Santa Cruz
Biotechnology (Santa Cruz, CA). p73 antibody (Ab-1) was obtained
from Oncogene Research Products (Boston, MA). Lipofectamine was
obtained from GIBCO (Grand Island, NY), and Fugene 6 was from Roche
(Indianapolis, IN). Reagents for the enhanced chemiluminescence system
and [3H]thymidine were obtained from Amersham (Arlington
Heights, IL). All other reagents were from Sigma (St. Louis, MO).
Cell culture and proliferation assay. Thoracic aortic rat A10 smooth muscle cells were obtained from American Type Culture Collection (Rockville, MD), maintained as described (34), and human aortic smooth muscle cells were obtained from Clonetics (San Diego, CA). The established tet-off clones were maintained with the addition of 0.25 µg/ml doxycycline, 350 µg/ml G418, and 250 µg/ml hygromycin and were used in experiments between subpassages 15 and 25. After the indicated times of doxycycline withdrawal, [3H]thymidine incorporation was assessed as previously described (34).
Stable transfection of tet-off constructs. The transfection protocol followed a method described by Yu et al. (38). A10 VSM cells were first transfected with tTA-IRES-Neo using lipofectamine and selected by the addition of G418 (350 µg/ml). The G418-resistant cells were cotransfected with linealized pBI-p73wt/EGFP and pTK-hygro plasmid DNA at a molar ratio of 5:1 using Fugene 6 and further selected with the addition of hygromycin B at a concentration of 250 µg/ml. Single clones were selected by GFP expression. These hygromycin B-resistant cells were further selected by withdrawal or by a series reduction of doxycycline concentration in culture medium from 125 to 62.5 ng/ml, as evidenced by GFP expression under fluorescent microscopy. The doxycycline-responding cells were evaluated for p73 overexpression by Western blot analysis and for cell proliferation by [3H]thymidine incorporation.
Western blots. Cells were grown to 80-90% confluence in six-well culture dishes. After withdrawal of doxycycline for the indicated times, cells were lysed, protein concentrations were determined by the Lowry method (Bio-Rad DC protein assay), and equal protein quantities were electrophoresed and Western blotted as previously described (34).
Balloon angioplasty. Lewis rats were anesthetized and the femoral artery was exposed. A balloon catheter was advanced by using a twisting motion until the balloon was under the inguinal ligament. Approximately 0.02 cc of saline was injected into the catheter to distend the balloon. The catheter was drawn back ~1 to 1.5 cm with constant pressure applied to the vessel. The pressure was then removed from the syringe and the catheter was reinserted into the artery, with a one-third turn (120°) of the catheter. The process was repeated for a total of three passes. At the end of the third ballooning, the catheter was removed so that only the tip of the catheter was occluding the vessel. After the indicated times, the rat was killed and the femoral artery was removed, lysed, and subjected to immunoblotting.
Evaluation of nuclear morphology. Cells were seeded in six-well culture dishes and treated as described. After removal of doxycycline for the times indicated (or no removal for controls), medium was aspirated and the cell culture dish was inverted over methanol for 10 min. The cells were then immersed in methanol for at least 10 min. Cells were stained in 1 µg/ml Hoechst 33258 in water with 1-2 g of nonfat dry milk. After being stained for 8-10 min, the cells were rinsed in water and completely dried. Nuclear morphology was visually evaluated by fluorescence microscopy.
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RESULTS |
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p73 is increased in arteries subjected to balloon angioplasty.
We have previously shown that p73, which is expressed at low levels in
serum-starved VSM cells consistent with reports in other normal tissues
(20), is increased after serum stimulation of VSM cells in
vitro, as well as in human atherosclerotic plaque tissue in vivo
(32). These data suggest that p73 is serving either a
promitogenic or growth-suppressive role in this tissue. To further
characterize the in vivo function of p73 in the rat tissue used in
subsequent experiments, we examined expression of this protein in VSM
cells from rat femoral arteries that had been damaged by balloon
angioplasty. Two weeks after Lewis rats were subjected to balloon
angioplasty, both the arteries subjected to angioplasty, as well as the
contralateral control femoral arteries, were harvested and the
homogenized tissue was electrophoresed and immunoblotted with p73
antibody. p73 in the arteries subjected to angioplasty was markedly
increased compared with the level of this protein in the contralateral
control arteries (Fig. 1), consistent
with our previous findings of increased p73 levels in human
atherosclerotic plaque tissue (32). This finding led us to
hypothesize that p73 is functioning in some manner in growth or
apoptotic control of the vasculature.
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Overexpression of p73 is independent of p53 but causes late
attenuation of p21 levels.
Although we have shown that p73 is increased in human plaque tissue and
in damaged rat arteries in vivo and in rat VSM cells stimulated with
serum in vitro, it is not clear whether induction of this protein has a
causal effect on proliferation of these cells, although it is tempting
to infer from these data that p73 is functioning either to increase
cell cycle transit or to limit cell proliferation. The former
possibility is supported by the finding that p73 is increased in
high-grade malignancies (6, 29) and the latter by reports
that p73 causes apoptosis in certain cancer cells
(28). To determine causality of p73 toward cell cycle
events in a normal VSM cell line, we utilized a tetracycline-inducible expression system to conditionally overexpress full-length p73. After a
tet-responsive VSM cell line had been successfully created (see
MATERIALS AND METHODS), these cells were stably
cotransfected with the wild-type p73 construct pBI-p73wt/EGFP obtained
from Dr. Vogelstein (38) and pTK-hygro, and doxycycline
withdrawal conditions were optimized in several clones to show maximal
induction of p73 protein levels compared with noninduced cells. After 1 day of doxycycline withdrawal, mild fluorescence was observed, and by 3 days of doxycycline withdrawal, strong fluorescence appeared and
persisted for at least 5 days. One clone was selected and used for
subsequent experiments. In initial experiments, doxycycline was
withdrawn for 5 days and expression of p73 was examined as a function
of doxycycline dose (Fig. 2). At
concentrations of doxycycline at 0.25 µg/ml or greater (including
concentrations down to 0.0625 µg/ml, not shown), p73 levels were
suppressed as assessed by immunoblotting, whereas, in the absence of
doxycycline, p73 was present at high levels. p53, which has
considerable sequence homology with p73, was not changed in cells
overexpressing p73 compared with those not overexpressing p73 at all
concentrations, including the absence of doxycycline
(concentration = 0) when p73 is at high levels (Fig. 2). The
tet-off-transfected cells were exquisitely sensitive to doxycycline,
such that even very low concentrations of this antibiotic (down to
0.0625 µg/ml) inhibited expression of the transfected p73 gene as
assessed by protein immunoblotting.
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Overexpression of p73 inhibits VSM cell cycle transit.
Because p73 overexpression has been shown to both correlate with
high-grade malignancies and to induce apoptosis in certain cancer cell lines, events that are difficult to reconcile with each
other, we next determined the effect of p73 overexpression on the VSM
cell cycle. Cell cycle transit of VSM tet-off cells grown continuously
in serum-containing medium and overexpressing p73 after 5 days of
doxycycline withdrawal (the time of maximal p73 overexpression)
was markedly inhibited in an inverse doxycycline dose-dependent
fashion, as measured by [3H]thymidine incorporation (Fig.
4). The finding that
[3H]thymidine incorporation was partially inhibited
despite minimal induction of p73 expression suggests that low levels of
expression of p73 protein (i.e., not seen on the Western blot at the
exposures used) are sufficient for cell cycle attenuation.
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p73 overexpression causes apoptosis.
Because inhibition of [3H]thymidine incorporation by
overexpressed p73 could result from a block in cell cycle transit
and/or cell death, we next asked when maximal apoptosis occurs
as a function of p73 overexpression. p73 induction of apoptosis
has been previously described in cancer cells (reviewed in Ref.
28), in some cases through superactivation of E2F in the
absence of p53 (9), but a death response to p73
overexpression in normal tissue or in vascular cells has not been
described. Doxycycline was withdrawn from the tet-off cells to increase
p73 expression for times ranging from 3 h to 5 days. The cells
were stained with Hoechst 33258 to assess apoptotic morphology and
subjected to fluorescence microscopy. Although there was minimal
nuclear condensation with up to 2 days of doxycycline withdrawal,
prominent apoptotic changes occurred after 5 days of doxycycline
withdrawal (Fig. 5A). To
confirm apoptosis in these p73-overexpressing cells, we
examined procaspase-3 cleavage, as another measure of
apoptosis, in the cells which showed maximal apoptosis
by Hoechst staining (5 days after doxycycline withdrawal). In this
experiment, the immunoblot shown in Fig. 3 was stripped and probed with
caspase-3 antibody. There was a high degree of procaspase-3 cleavage,
as indicated by a decrease of the 34-kDa band and an increase in the
20-kDa protein, only in the cells in which doxycycline had been
withdrawn for 5 days (Fig. 5B), consistent with the Hoechst
staining (see Fig. 5A). Thus apoptosis is a late
phenomenon after p73 overexpression, and maximal apoptosis occurs at the time of maximal p73 overexpression independently of p53
changes (compare Figs. 5 and 3). Furthermore, maximal apoptosis also corresponds to p21 attenuation, further supporting p21's survival
function in these cells.
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DISCUSSION |
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Although the tumor-suppressor protein p53 has been extensively characterized due to the fact that it is mutated in the majority of human tumors, the function of its homolog p73 has been considerably more elusive. Various forms of p73 have been shown to be proapoptotic and growth suppressive (9, 11, 17, 39) or antiapoptotic (21), but this protein has been studied principally in malignant tissue, likely due to the findings of very low p73 expression in most normal tissues under basal conditions (13). Consistent with its putative, although elusive, role in cancer biology, p73 knockout mice were generated and had been expected to develop spontaneous malignancies similar to p53 knockout mice. However, p73 knockout mice do not show spontaneous tumors (37), and, despite the sequence similarity between p73 and p53, p73 mutations have been found, although infrequently, in human tumors (18). These data are difficult to reconcile with findings that p73 is overexpressed in certain cancers and that the level of its expression correlates with aggressiveness of these tumors. What the data do confirm is that the role of p73 in growth regulation, while likely profound, is not well understood.
Due to its original serendipitous discovery in chromosomal region that is frequently deleted in neuroblastoma (12), p73 studies have been largely confined to the field of cancer biology. Naturally, considerable excitement in the neurological cancer community was generated because of the idea, which turned out to be an oversimplification, that p73 may be a p53 homolog, the deletion of which is pathogenic for neural cancers. A study of 51 different normal tissues showed very low p73 expression (13), but there is no information on growth-stimulated p73 expression in any normal tissue. The evolving role of p53 in vascular lesions, where its inhibition has been postulated to contribute to the development of coronary restenosis (27), led to our study of p73 in this tissue. Our finding that overexpression of p73 leads to apoptotic changes in VSM cells opens up a new area of investigation of this protein as a potential therapeutic signaling protein, in which this protein could ultimately prove useful in a clinical setting to limit aberrant the proliferation of VSM cells.
p53 is increased after balloon angioplasty of rabbit arteries
(1) in a manner that parallels apoptosis
(25). The first (and thus far, only) published description
of p73 in VSM cells was made in our laboratory, where the level of
expression of p73 was shown to increase in serum-stimulated cells as
well as in vivo in human atherosclerotic plaque tissue
(32). Our finding in the present study that p73
overexpression causes apoptosis in VSM cells is an example of
the similarity between the tumor-suppressor protein p53 and its homolog
p73, yet the association of p73 with a decrease in p21 level at the
time of maximal apoptosis is surprising and represents another
significant divergence between p73 and p53 signaling. These data also
suggest that p73 may utilize the survival or assembly factor function
of p21 (2, 5, 15, 24, 33), a function that has not been
demonstrated for p53. Interestingly, a similar, slight decrease in p21
was recently reported in cells in which p73 was overexpressed
(3), but the authors of that study did not discuss this finding.
What is p73 doing in the vasculature? It has been well established that, after endothelial injury, it is the proliferation of VSM cells that contributes to the progression of the atherosclerotic state (22). Furthermore, the VSM cell proliferative response after coronary angioplasty is similar to the healing response of other tissues to injury, but in 25-50% of patients this response leads to coronary restenosis. A similar processes is believed to underlie hemodialysis vascular access stenosis. Based on our data in this study, we believe it is possible that p73 functions in vivo to attenuate the exuberant response of VSM cells to vascular injury. The proapoptotic action of p73 that we observed in VSM cells may parallel the action of p53 in cancer (1), yet the mechanism utilized by the two homologs in VSM cells may be disparate, involving p21 suppression in the case of p73. The findings that p53 and p73 knockout mice display quite disparate phenotypic abnormalities in terms of cancer susceptibility prove that different pathways are activated by these homologous proteins. Furthermore, p73 and p53 have been described as having competing effects (31, 36), such that the downstream response of these transcription activators may be measured in their integrated effects on p21 or other cyclin-associated proteins.
The provocative possibility of the existence of a link between p73 and atherosclerosis may be seen in the finding that p73 maps to chromosome 1p36 (12), whereas a candidate human atherosclerosis susceptibility locus is present at 1p36-32 (35). A clinical connection between these two events could conceivably result from a loss of a normal apoptotic pathway associated with overexuberant VSM cell proliferation in response to atherosclerotic stimuli or tissue injury (as would occur during angioplasty), leading to the atherosclerotic condition. In addition, now that atherosclerosis is known to involve inflammatory mediators (23), the severe inflammatory response seen in the p73(-/-) mice (37) is consistent with our data that the overexpression of p73 both decreases VSM cell proliferation and causes apoptosis, processes that can be described as anti-inflammatory. The connection between p73 and atherosclerosis, of course, begs further study.
Because VSM cell proliferation is the pathology underlying failure of angioplasty procedures, manipulation of p73 suggests possibilities for therapeutic intervention. Although p73 mutations have not been observed in human tumors, the possibility that decreased levels or mutation of p73 underlies atherosclerotic or other vascular proliferative diseases is provocative. Furthermore, supporting this gene as a potential gene therapeutic agent, p73 is assumed to replace or enhance the p53 protective role in p53-lacking cells (7), and there are now serious concerns regarding augmentation of p53 wild-type activity in mice (30). Thus p73 deserves further study as a tool to prevent VSM cell proliferation under conditions leading to atherosclerosis and other vascular diseases.
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ACKNOWLEDGEMENTS |
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We thank Dr. Clare Gregory for assistance with the balloon angioplasty experiments.
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FOOTNOTES |
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This work was supported by grant 1R21CA 91259-01A1 from National Cancer Institute, the Research Service of the U.S. Department of Veterans Affairs, a University of California Davis Health System Award, and a grant from the National Kidney Foundation.
Address for reprint requests and other correspondence: R. H. Weiss, Div. of Nephrology, TB 136, Dept. of Internal Medicine, Univ. of California, Davis, CA 95616 (E-mail: rhweiss{at}ucdavis.edu).
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.
10.1152/ajpcell.00211.2002
Received 10 May 2002; accepted in final form 9 September 2002.
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