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Originally published In Press as doi:10.1074/jbc.M101188200 on May 3, 2001

J. Biol. Chem., Vol. 276, Issue 28, 26180-26188, July 13, 2001
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Lithium Inhibits Cell Cycle Progression and Induces Stabilization of p53 in Bovine Aortic Endothelial Cells*

Catherine D. MaoDagger, Phuong Hoang, and Paul E. DiCorleto

From the Department of Cell Biology, The Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195

Received for publication, February 7, 2001, and in revised form, April 30, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Lithium affects development of various organisms and cell fate through the inhibition of glycogen synthase kinase-3beta and induction of the Wnt/beta -catenin signaling pathway. In this study, we investigated the effects of lithium on primary bovine aortic endothelial cells (BAEC). Lithium treatment of BAEC induced beta -catenin stabilization but failed to activate the transcriptional activity of the beta -catenin/T-cell factor complex. Lithium caused a sustained G2/M cell cycle arrest without affecting cell viability. Reversibility of this cell cycle arrest occurred up to 3 days after treatment but was reduced thereafter. Lithium-treated BAEC exhibited a senescent-like morphology with an increase in cells positive for the senescence-associated-beta -galactosidase activity. Lithium also increased the expression of p21Cip, a cyclin-dependent kinase inhibitor, both at the protein and RNA levels. No change in p21Cip mRNA stability was observed, whereas the transcriptional activity of a p21Cip promoter-luciferase construct containing p53 binding sites was increased after lithium treatment. Furthermore, lithium caused increased transcription of a reporter gene under the control of a promoter containing the p53 consensus binding sites both in transiently transfected BAEC and in a stably transfected fibroblast cell line. Lithium caused accumulation of p53 protein in BAEC without affecting p53 mRNA levels. Finally, up-regulation of p21Cip in response to lithium did not occur in mouse embryonic fibroblasts that were null for p53 alleles, confirming the dependence on a p53 pathway for this lithium effect. These findings demonstrate for the first time that lithium induces also stabilization of the tumor suppressor p53 and reveal a new mechanism that may contribute to the neuroprotective effects of lithium.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Lithium has profound effects on the embryonic development and patterning in various organisms including Dictyostelium and Xenopus (1). Inhibition of the development of the yolk sac vasculature by lithium has been observed in the chick embryo (2). Lithium is also the main therapeutic agent for the treatment of patient suffering from bipolar disorder (3). To date, the underlying mechanisms and the molecular targets of lithium action have not been established neither during development nor during therapy.

The developmental phenotype that is induced by lithium in Xenopus is similar to that caused by alteration in the expression of Wnt proteins (1, 4, 5) and of Wnt signaling components such as beta -catenin (6) and the transcription factor T-cell factor (TCF)1 (1, 7). Lithium has been shown to be a specific and noncompetitive inhibitor of glycogen synthase kinase-3beta (GSK-3beta ) activity in vitro (8, 9) and in vivo (10). GSK-3beta is a serine/threonine kinase that controls cell survival and cell fate through its involvement in multiple signaling pathways (11). Based on these findings, lithium is commonly used both as a potent mimetic of Wnt signaling and as a specific inhibitor of GSK-3beta (1, 5-11). beta -Catenin, both a scaffolding protein in cadherin-mediated cell adhesion and a signaling molecule in the Wnt signaling pathway, is a target of GSK-3beta (12, 13). In the absence of a stimulatory signal, low levels of beta -catenin are tightly regulated via phosphorylation by GSK-3beta . This represents a signal for beta -catenin degradation via the ubiquitin-dependent pathway (12, 13). Inactivation of GSK-3beta by Wnt signaling or by lithium leads to stabilization and nuclear translocation of beta -catenin where it associates with TCF transcription factor members (14) and activates transcription of genes involved in cell adhesion (15) and cell proliferation (16, 17).

Abnormalities in the regulation of beta -catenin have been implicated in colorectal and melanoma tumorigenesis (12, 13). However, in vivo overexpression of an N-terminally truncated form of beta -catenin in intestinal epithelium did not increase tumorigenesis but increased both proliferation and apoptosis of undifferentiated cells without inducing cell differentiation (18). The proliferation of terminally differentiated cells within the same intestinal crypt was not affected (18). Similarly, only the proliferation of progenitor cells within the intestinal crypt was impaired in mice null for the transcription factor TCF4 alleles (19). Overexpression of beta -catenin, either wild-type or an S37A mutant, in different cell backgrounds yielded various results. Proliferation of the epithelial Madin-Darby canine kidney cells has been observed (20), whereas apoptosis appeared to be the major effect in Drosophila retinal neurons (21), in mouse neurons (22), and in fibroblasts NIH3T3 (23). In the last, the apoptotic effects of beta -catenin appeared independent of its transactivation function and interaction with TCF (23).

Endothelial cells of the adult vasculature are characterized by a quiescent state, which is controlled in part by the organization of cadherin and catenin complexes at cell-cell contacts (24). However, in response to injury or cytokine stimulation, tyrosine phosphorylation of beta -catenin and dissociation from the cadherin complex is associated with EC migration and proliferation (24). Conversely, sequestration of beta -catenin by the endothelium-specific VE-cadherin in the junctional complex inhibits EC migration and proliferation (25). beta -Catenin cleavage by caspase-3 and dissociation from the complex is also an early event in the EC onset to apoptosis (26). Truncation of the beta -catenin binding domain of VE-cadherin induces EC apoptosis, indicating that beta -catenin may have a survival role in EC (27).

Increased cytoplasmic levels of beta -catenin have been reported in EC during the neovascularization process occurring after myocardial infarction (28). The expression of different components of the Wnt signaling pathway in vascular cells is also altered in a rat model of restenosis (29) or in vitro in response to mitogens (29) or cell passage (30). EC proliferation occurs during angiogenesis and various vascular diseases (31). Cell cycle progression in EC is inhibited, as in other mammalian cells, by two key regulators: the cyclin-dependent kinase inhibitor p21Cip and the tumor suppressor p53 (32). Increased expression of p21Cip and p53 is observed in atherosclerotic lesions (31). Stabilization of p53 and induction of p21Cip by a variety of injuries, stresses, or inhibitors of proliferation lead to cell cycle arrest either at the G1/S or G2/M transitions (32).

Lithium has been shown to affect cell proliferation either positively (33) or negatively (34), depending on the cell type. Lithium induces proliferation of erythroid progenitor cells (35) and terminal differentiation of WEHI-3B myelomonocytic leukemia cells (36). Wnt5a and Wnt10b are also able to promote proliferation of hematopoietic progenitor cells (37). Lithium, similarly to Wnt7A, has been shown to extend neurite outgrowth and to induce differentiation of cerebellar granule neurons (38). Several recent reports suggest that lithium may exert its therapeutic effects through neuroprotection against various stresses: ischemia, glutamate excitotoxicity, radiation, and heat shock (39-42). Such survival effects of lithium on neuronal cells seem to be mediated in part by the activation of the survival Akt/protein kinase B pathway (40) as well as by the inactivation of GSK-3beta activity (42).

Taken together, it appears that lithium as well as Wnt and beta -catenin signaling may have various effects on cells depending on their differentiated state and whether they are immortalized or transformed. In the current report, we have determined that in primary EC lithium induces stabilization of both beta -catenin and the tumor suppressor p53. This precedes cell cycle arrest in G2/M phase. Thus, our results reveal a novel mechanism that may account for the developmental and neuroprotective effects of lithium.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cells, Treatments, and Reagents-- Bovine aortic EC (BAEC) were isolated as described previously (43) and grown in Dulbecco's modified Eagle's medium/F-12 containing 5% fetal bovine serum and used at passages 3-8. At least four different isolates of BAEC were used in the present study. Mouse fibroblast (12)1/CA cells carrying a p53-responsive beta -galactosidase reporter construct have been described (44). The mouse embryonic fibroblasts MEFp53+/+, MEFp53+/-, and MEFp53-/- were derived from parental, heterozygous, and p53 knockout mice, respectively, and were kindly provided by Dr. Tyler Jacks (45). All of the mouse fibroblast cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Life Technologies, Inc.). Cell irradiation with 25 J/m2 UV light for 10 s was used as positive control for p53 activation and was conducted as previously described (44). Actinomycin D was from Sigma.

Measurement of [3H]Thymidine Incorporation and [3H]Leucine Incorporation-- Cells were grown in 24-well culture clusters for 24 h prior to treatment with LiCl, or NaCl as control, with the indicated doses and times. In the last 2 h of treatment, 1 µCi/ml [methyl-3H]thymidine (specific radioactivity 6.7 Ci/mmol; ICN) was added. Thereafter, the cells were washed rapidly with ice-cold 5% trichloroacetic acid and then incubated on ice for 20 min in 5% trichloroacetic acid. After two washes with ice-cold PBS, the cells were solubilized with 0.25 N NaOH and transferred to counting vials containing 5 ml of liquid scintillant (ICN). All results represent triplicate samples and three independent experiments. For the determination of [3H]leucine incorporation, 24 h after seeding, the cells were treated with various doses of NaCl or LiCl for 24 h prior to the addition of 0.5 µCi/ml of [3H]leucine (specific radioactivity 132 Ci/mmol; ICN) followed by a 24-h incubation at 37 °C. Thereafter, the cells were washed and solubilized as described above. The experiment was carried out with triplicate samples and was repeated twice.

Cell Cycle Analysis-- The cell cycle distribution was determined by flow cytometric analysis of propidium iodide-labeled cells. Briefly, BAEC were plated in six-well culture clusters 24 h prior to treatment with either NaCl or LiCl for the indicated doses and times. After trypsinization, cells were collected, washed twice with ice-cold PBS, and fixed in ice-cold 70% ethanol. Cells were then washed twice with ice-cold PBS, resuspended in PBS containing 100 units/ml RNase A, incubated at 37 °C for 30 min, stained with propidium iodide (20 µg/ml), and analyzed by FACScan (Becton Dickinson).

Senescence-associated beta -Galactosidase Assay-- BAEC were grown on glass coverslips in six-well culture clusters 24 h prior to cell treatment either with NaCl or LiCl. Four days post-treatment, BAEC were stained for senescence-associated beta -galactosidase (SA-beta -galactosidase) activity at pH 6 for 16 h, as previously described (46). Phase-contrast and color photomicrographs of stained cells were taken.

Northern Blot Analysis-- Total RNA was isolated by the acid guanidine thiocyanate/phenol/chloroform method using Trizol (Life Technologies). RNA (10-20 µg) was size-fractionated in a 1% agarose gel containing 6% formaldehyde in MOPS buffer and then transferred to Nytran nylon membrane (Schleicher and Schuell) using 10× SSC (1.5 M NaCl and 0.15 M sodium citrate). The filters were hybridized for 12-16 h at 65 °C with 32P-labeled DNA probes (106 µCi/ml) in Church and Guilbert buffer. DNA probes were labeled with [alpha -32P]dCTP (specific activity 3000 Ci/mmol; ICN) using the oligoprimer labeling kit (Amersham Pharmacia Biotech). After hybridization, the membranes were washed under standard conditions and autoradiographed using X-Omat MR films (Eastman Kodak Co.). Radioactive mRNA signals were quantified by PhosphorImager (Molecular Dynamics, Inc.). Each experiment was repeated 2-3 times.

The DNA probes used in this study were as follows: full-length human p21Cip cDNA (47), full-length human p53 cDNA (48), and full-length mouse cyclin D1 (49). The probes for p27Kip and rpL32 were generated by reverse transcriptase-polymerase chain reaction using the following primers: for human p27Kip, primer pair 5'-GAAAGATGTCAAACGTGCG-3' and 5'-GAGCTGTTTACGTTTGAC-3', generating a 600-base pair product; for human rpL32, primer set 5'-GCCAGATCTTGATGCCCAAC-3' and 5'-CGTGCACATGAGCTGCCTAC-3', generating a 280-base pair product.

Cell Fractionation and Extracts-- Whole cell extracts were prepared as follows. After incubation and treatments, cells were washed with PBS and lysed in 10 mM Hepes, pH 7.6, 1.5 mM MgCl2, 1 mM EDTA, 1% Triton X-100, 10 mM NaF, 2 mM NaVO3, 1 mM dithiothreitol supplemented with a mixture of protease inhibitors (Roche Molecular Biochemicals). For cell fractionation into cytosolic and membrane extracts, cells were washed in PBS, collected, and disrupted in 10 mM Hepes, pH 7.6, 1.5 mM MgCl2, 1 mM EDTA, 10 mM NaF, 2 mM NaVO3, 1 mM dithiothreitol, and protease inhibitors by repeated passage through a 23-gauge needle. After removal of nuclei by centrifugation at 800 × g for 5 min, the supernatant was centrifuged at high speed (100,000 × g for 30 min) to separate the membrane (pellet) and the cytosolic (supernatant) fractions. Nuclear extracts were prepared as previously described (50).

Immunoblot Analysis-- Protein (30 µg) was subjected to SDS-polyacrylamide gel electrophoresis (10%) and then transferred to polyvinylidene difluoride membrane (Millipore Corp.). Loading of equal amounts of protein was verified by staining with Ponceau-S (Sigma). Filters were incubated for 2 h at room temperature in 5% nonfat milk blocking mix (Bio-Rad) in 50 mM Tris-HCl, pH 7.6, 0.15 M NaCl, 0.05% Tween 20, followed by 12-16 h at 4 °C with either mouse monoclonal anti-p53 antibody (PAb240) or rabbit polyclonal anti-p27Kip antibodies (Santa Cruz) or rabbit polyclonal anti-beta -catenin (Sigma) in the same buffer. After 3 washes, blots were incubated with the appropriate secondary antibody conjugated to horseradish peroxidase Upstate Biotechnologies, Inc., Lake Placid, NY, developed using the enhanced chemiluminescence substrate (Amersham Pharmacia Biotech), and exposed to Kodak X-Omat-MR film. The polyclonal antibodies specific for p21Cip used in this study were directly conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Inc.).

Transient Transfection and Luciferase Assay-- BAEC were grown in 12-well culture clusters, and 24 h later at 60% confluency, cells were transfected for 2 h in serum-free medium with 5 µl of Exgen 500 (Euromedex) and with 0.5 µg of the following cesium chloride-purified luciferase constructs: the TOPflash and FOPflash constructs containing, respectively, wild-type or mutated TCF binding sites upstream of the thymidine kinase minimal promoter (14); the p21 promoter luciferase constructs -2300-p21L, -210-p21L, and -94-p21L previously described (48); the p53-CA-luciferase reporter construct containing p53 consensus A sites and HSP70 minimal promoter (51); and the promoterless pGL3-basic (Promega). 5 ng of pRSV-beta -galactosidase was co-transfected to monitor the efficiency of transfection. Normal medium was then added for 22 h prior to treatment with NaCl, LiCl, or UV light as positive control for the indicated times. Cells were lysed 30-48 h after transfection, and the luciferase activity assays and beta -galactosidase activity assays were performed accordingly to the manufacturers' instructions (Promega and Tropix, respectively) and monitored using a Dynex luminometer.

Statistical Analysis-- Data are presented as the mean ± S.D. of at least three independent experiments unless designated otherwise. Statistical analysis was performed using Student's t test, and a value of p < 0.05 was considered to be significant.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Lithium Induces beta -Catenin Stabilization in BAEC without Inducing an Active beta -Catenin-TCF Pathway-- We investigated the effect of lithium on the beta -catenin-TCF pathway in primary BAEC by using two standard assays: stabilization and nuclear accumulation of beta -catenin and transcriptional activation of a luciferase reporter driven by TCF-responsive elements. The epithelial HEK293 cells were used as positive control, since they are responsive to both lithium and Wnt signaling (52). As shown in Fig. 1A, lithium treatment for 12 h in BAEC increased the cytosolic pool of beta -catenin, similar to the case in epithelial HEK293 cells, a known responsive cell line for Wnt signaling. A slight increase of beta -catenin nuclear pool was also observed in both cell lines. To test whether this beta -catenin stabilization was followed by an increase in the beta -catenin-TCF complex activity, BAEC and HEK293 were transfected with either the TOPflash or FOPflash constructs (14). 24 h post-transfection, the cells were treated for 12 h with either 5 mM NaCl as control or with 5 mM LiCl. A 4-fold increase in luciferase activity was observed in HEK293 cells transfected with TOPflash and treated with lithium but not with the FOPflash construct containing mutated TCF sites (Fig. 1B). However, under the same conditions, lithium did not increase the activity of the TOPflash construct in BAEC. This lack of effect in BAEC was also observed with higher doses of LiCl (20 mM) (not shown). Therefore, in contrast to HEK293, lithium induced in BAEC stabilization of beta -catenin without inducing an active beta -catenin-TCF complex.


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  		Fig. 1.   Lithium induces beta -catenin stabilization but not beta -catenin-TCF complex transcriptional activity. A, stabilization of beta -catenin in the different cell extracts was followed by Western blot analysis. BAEC and HEK 293 cells were treated with 5 mM NaCl (-) as control or 5 mM LiCl (+) for 12 h prior to cell fractionation. Equal amounts of protein were loaded on the gel and verified by Ponceau S staining of the membrane. The membranes were probed with specific polyclonal antibodies anti beta -catenin, goat anti-rabbit horseradish peroxidase, and ECL reagents. B, determination of the transcriptional activity of beta -catenin-TCF complex. BAEC and HEK293 cells were co-transfected with 0.5 µg of either the TOPflash (TOP) or FOPflash (FOP) luciferase reporter constructs containing wild-type and mutated TCF binding sites, respectively, and 5 ng of pRSV-beta -galactosidase for normalization of transfection efficiency. 24 h post-transfection, cells were treated for 12 h with either 5 mM NaCl as control or 5 mM LiCl. Luciferase activities were determined and normalized for beta -galactosidase activities for each condition. The presented result (mean + S.D. of duplicates) is representative of five independent experiments.

Lithium Inhibits Proliferation of Primary BAEC-- The effect of lithium on BAEC proliferation was examined by measuring the incorporation of [3H]thymidine into DNA. Subconfluent and asynchronous BAEC were incubated with various concentrations of LiCl or with NaCl as a control for the indicated times. Cells were pulse-labeled with [3H]thymidine in the last 2 h of the incubation. As shown in Fig. 2A, the amount of [3H]thymidine incorporation in lithium-treated cells (5 mM) was only 49.7 ± 7.9% at 12 h and 19.5 ± 7.9% at 24 h as compared with sodium-treated cells (p < 0.05). This effect was also observed at lower concentration of lithium (3 mM) with an inhibition of 32 ± 5.8 and 43 ± 7.9% after, respectively, 24- and 48-h treatment (p < 0.05). Cell viability was monitored by measuring the rate of protein synthesis (incorporation of [3H]leucine into proteins) and the number of cells was determined in a parallel experiment. As shown in Fig. 2B, the number of BAEC was decreased by 38 ± 2.5 and 55 ± 2.45% after 48-h treatment of a 70% confluent culture with 5 and 10 mM lithium, respectively. After correcting for cell number, the protein synthetic rate was found to be unchanged after 48 h of lithium treatment with doses up to 10 mM (Fig. 2C). These results indicated that lithium inhibits BAEC proliferation without affecting their viability.


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  		Fig. 2.   Lithium inhibits BAEC proliferation. A, determination of DNA synthesis in BAEC treated with LiCl or NaCl for the indicated doses and times by measuring [3H]thymidine incorporation into DNA. Cells were pulse-labeled with 1 µCi/ml [3H]thymidine in the last 2 h of treatment, and trichloroacetic acid-precipitable radioactivity was counted. The results are expressed as percentage of the values obtained for the control-treated cells (100%) and means ± S.D. of three independent experiments are reported. B, determination of cell viability by incorporation of [3H]leucine into protein. BAEC (105) were left untreated for 24 h and were then treated for 24 h with LiCl or NaCl prior to the addition of 0.5 µCi/ml [3H]leucine and a further 24 h incubation. Trichloroacetic acid-precipitable radioactivity was measured by scintillation counting. The number of cells under each condition was determined in parallel.

Lithium Induces a G2/M Cell Cycle Arrest in BAEC-- Inhibition of cell proliferation can occur through activation of several possible checkpoints during cell cycle progression. To determine the effects of lithium on the cell cycle, subconfluent and asynchronous BAEC were treated with LiCl for various times, and cell cycle profiles were monitored by flow cytometric analysis of DNA content (Fig. 3A and Table I). The number of cells with a 4N DNA content was increased at 16 and 24 h in a dose-dependent manner by lithium (Fig. 3A and Table I). A greater effect was observed with 10 mM LiCl for 24 h with 35 ± 4.1% of cells in the G2/M phase of the cell cycle as compared with 20.1 ± 5.6% for 5 mM LiCl-treated cells or with 10% for the control cells (p < 0.05). Since lithium is known also to inhibit the inositol 1,4,5-trisphosphate breakdown in myoinositol, we investigated whether the effect of lithium occurred by inhibiting the phosphoinositol turnover. The addition of 10 mM myoinositol, the limiting factor in the inositol pathway turnover, did not affect the ability of lithium to induce a cell cycle arrest in BAEC (Fig. 3A), thus ruling out that the cell cycle arrest induced by lithium occurs via depletion of the inositol pathway. The percentage of cells with less than 2N DNA content, characteristic of apoptotic cells, was not modified by lithium treatment (not shown). The accumulation of cells in G2/M phase persisted over 4 days (Table I). After 24 h of treatment of BAEC, lithium removal resulted in an increase of cells in the S phase (22 ± 6.4%) in 24 h similar to the percentage observed in control cells (29.6 ± 6.1%) (p > 0.1). After 4 days of treatment, only a slight increase in S phase cells was observed after lithium removal (p < 0.05) (Table I). To confirm the loss of the reversibility of lithium-induced cell cycle arrest, BAEC pretreated with sodium or lithium for 3 days were seeded at 5 × 105 cell density in the absence of lithium. After 2 days of culture, the number of lithium-pretreated cells was reduced by about 30-40% as compared with sodium-pretreated cells, whereas there was no significant difference at 24 h, indicating that the adhesion ability of lithium-pretreated cells was not impaired (Fig. 3B). Therefore, these results were also indicating that lithium-induced cell cycle arrest in BAEC occurred in the absence of cytotoxicity.


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  		Fig. 3.   Lithium induces a G2/M cell cycle arrest in BAEC. A, cell cycle analysis of subconfluent (70-80%) and asynchronous BAEC treated with either 5 mM LiCl, 10 mM LiCl, or 10 mM NaCl for 16 and 24 h. At 24 h, a set of cells was also set up with 10 mM myoinositol. After trypsinization, the cells were ethanol-fixed and subjected to propidium iodide staining for quantification of DNA content by flow cytometry. The percentage of cells in each phase of the cell cycle was determined. Representative flow cytometric analyses of BAEC cell cycle after lithium treatment of 3-5 independent experiments are shown. B, determination of the ability of BAEC pretreated with lithium to reenter the cell cycle. Subconfluent and asynchronous BAEC were pretreated with either 5 mM NaCl (-) or with 5 mM LiCl (+) for 72 h prior to trypsinization. 5 × 104 pretreated cells were then seeded, and the number of cells after 24 or 48 h of culture was determined. Results are means ± S.D. of two independent experiments done in duplicate.

                              
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Table I
Lithium affects cell cycle progression in BAEC
BAEC were treated with either 10 mM NaCl as control or with LiCl (1-10 mM) from 16 h up to 4 days. Reversibility of cell cycle arrest was tested in BAEC pretreated for 24 h or 3 days with either NaCl as control or 5 mM LiCl and then washed twice with normal medium for lithium removal and further incubated in normal medium for 24 h (+ 24 h R). Cell cycle analysis was done by flow cytometry of DNA content after propidium iodide staining. The percentage of cells in each phase of the cell cycle was determined, and the results are reported as mean ± S.D.

Lithium Induces a Senescence-like Phenotype in BAEC-- Morphological changes in BAEC were observed after lithium treatment with an increased size and a more spread and flattened appearance (Fig. 4A). No rounded cells, characteristic of apoptosis, were observed. The number of multinucleated cells was slighty increased, but after cell nuclei staining with 4',6-diamidino-2-phenylindole (DAPI), the most striking features of lithium-treated BAEC were enlarged nuclei and uncondensed chromatin, characteristic of interphase nuclei (Fig. 4B). Altogether, these observations were consistent with cell cycle arrest occurring in early G2. These morphological changes and the irreversibility of the cell cycle arrest (Fig. 3B and Table I) had similarity to the senescent cells (53). Therefore, we analyzed lithium-treated BAEC for expression of the SA-beta -galactosidase marker (46). The beta -galactosidase staining was more intense in lithium-treated cells as compared with control cells (Fig. 4C). After counting the positive cells among 100 cells total, a 4-fold increase in SA-beta -galactosidase-positive cells after 5 days of lithium treatment was observed as compared with sodium-treated cells.


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  		Fig. 4.   Lithium induces a senescent-like morphology in BAEC. A, phase-contrast photomicrograph (× 10 magnification) of live BAEC treated for 5 days with either 5 mM LiCl or 5 mM NaCl. B, fluorescence photomicrograph (× 20 magnification) of BAEC treated with either 5 mM NaCl or 5 mM LiCl for 24 h and then fixed and stained with beta -catenin (green) and with 4',6-diamidino-2-phenylindole (DAPI) for nuclei visualization (blue). C, phase-contrast and color photograph (× 20 magnification) of BAEC stained for the SA-beta -galactosidase activity (blue). BAEC were grown on coverslips and at 70% confluence were treated with either 5 mM LiCl or 5 mM NaCl for 5 days. SA-beta -galactosidase staining was done at pH 6.

Lithium Up-regulates the Expression of p21Cip in BAEC-- To further characterize the effects of lithium on BAEC, we investigated the expression of genes involved in the control of cell cycle progression. As shown in Fig. 5A, lithium treatment induced a time-dependent increase in the cyclin-dependent kinase inhibitor p21Cip mRNA levels, with a maximum 4-fold increase after treatment with 5 mM LiCl for 8 h. This increase was sustained up to 48 h as detected by Northern blot. Meanwhile, the expression of p27Kip, another cyclin-dependent kinase inhibitor, was slightly down-regulated (1.5-fold decrease at 4 and 8 h). The expression of cyclin D1, a target gene of the beta -catenin-TCF pathway (16), was biphasic with a slight increase of 1.5-fold at 4 and 8 h of lithium treatment followed by a down-regulation at 24 and 48 h as compared with control cells. The mRNA levels of the tumor suppressor p53 was not significantly modified by lithium treatment (Fig. 5A). Up-regulation of p21Cip expression was also confirmed at the protein level by Western blot analysis. A time-dependent accumulation of p21Cip protein was observed in whole cell extracts of BAEC treated with 5 or 10 mM LiCl as compared with control-treated cells (Fig. 5B). On the other hand, the expression of p27Kip protein was down-regulated by lithium.


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  		Fig. 5.   Lithium increases p21Cip expression. A, Northern blot analysis of cell cycle-regulated genes in BAEC after lithium treatment. Subconfluent BAEC were incubated with NaCl (-) or LiCl (+) for the indicated times. Total RNAs were isolated and then analyzed by Northern blot for the expression of cyclin D1, p21Cip, p27Kip, and p53 mRNAs. Intensities of the radioactive bands were quantified by a PhosphorImager. Normalization of the RNA amounts was achieved using the expression of the ribosomal protein L32 (rpL32) mRNA as standard. B, Western blot analysis of p21Cip and p27Kip protein expression in BAEC after lithium treatment. Whole cell extracts from BAEC treated with 10 mM NaCl (0) or with LiCl for the indicated doses and times were analyzed by immunoblotting with specific antibodies for p21Cip and p27Kip.

Expression of p21Cip Is Transcriptionally Regulated by Lithium-- We investigated next whether an increase of p21Cip gene transcription or an increase of p21Cip mRNA stability could account for lithium-induced p21Cip mRNA expression in BAEC. The p21Cip mRNA stability was determined in BAEC after exposure to 5 mM NaCl or 5 mM LiCl for 6 h followed by an incubation with actinomycin D, an inhibitor of RNA synthesis, for various times. A similar half-life of about 3.5 h was determined for p21Cip mRNA both in control and lithium-treated cells, thus ruling out a post-transcriptional effect (Fig. 6A). The transcriptional activation of p21Cip promoter by lithium was investigated using the luciferase reporter driven by different fragments of the human p21Cip promoter (48) for which binding sites for various transcription factors have been delineated. These results are represented schematically in Fig. 6B. After 12 h of treatment with 10 mM LiCl, no significant changes in luciferase activities were observed with the smaller fragments, -291/+16 and -94/+16, of the p21Cip promoter (Fig. 6B). In contrast, a 1.7-fold induction of luciferase activity was observed with the -2300/+16 fragment of p21Cip promoter, which contains two binding sites for p53 (p < 00.5) (Fig. 6B). Altogether, these results showed that lithium regulated the transcription of p21Cip gene and suggested that p53, a potent inducer of p21Cip expression (54), may be involved in this process.


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  		Fig. 6.   Transcriptional regulation of p21Cip expression by lithium. A, determination of p21Cip mRNA stability in BAEC after lithium treatment. BAEC were treated with 5 mM NaCl or 5 mM LiCl for 12 h prior to the addition of 10 µg/ml actinomycin D for the indicated times. p21Cip mRNA expression was followed by Northern blot analysis. Intensities of the radioactive bands were quantified by PhosphorImager and plotted versus time of actinomycin D treatment, and the p21Cip mRNA half-life was determined in the absence or presence of lithium treatment. B, transcriptional activation of p21Cip promoter by lithium in BAEC. BAEC were transfected with 0.5 µg of a luciferase reporter driven by the fragment -2300/+16 (-2300-p21L), -291/+16 (-291-p21L), or -94/+16 (-94-p21L) of the p21 promoter sequence. The promoterless luciferase construct pGL3 basic was used as further control, and co-transfection with 5 ng of pRSV-beta gal vector was done for normalization of transfection efficiency. 24 h post-transfection, BAEC were treated with either 10 mM NaCl or LiCl for 12 h. Luciferase activities were measured in the cell lysates and normalized with beta -galactosidase activities. Fold induction represents the ratio of the values obtained in lithium-treated versus sodium-treated cells for each construct after normalization. Results are mean ± S.D. of six independent experiments performed in duplicate. *, p < 0.05 significantly different from the control.

Lithium Activates and Stabilizes Endogenous p53-- We next examined whether the transcriptional activity of p53 could be increased by lithium. First, the mouse (12)1/CA fibroblast carrying consensus response elements for p53, controlling expression of the beta -galactosidase reporter gene (44), were treated either with NaCl, LiCl, or UV light as a positive control. Whole cell extracts were prepared at various times after treatment, beta -galactosidase activity was determined, and expression of p21Cip was followed by immunoblotting. Maximal effects were observed at 16 h after lithium addition, with a reproducible 2.5-fold increase in beta -galactosidase activity as compared with sodium-treated cells (Fig. 7A). UV light irradiation induced beta -galactosidase activity by about 15-fold, as previously reported (44). A similar accumulation of p21Cip protein was observed both in lithium-treated and UV-irradiated (12)1/CA cells (Fig. 7A).


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  		Fig. 7.   Lithium induces p53 activation and accumulation. A, lithium-induced activation of p53 in (12)1/CA fibroblasts. The mouse fibroblast line (12)1/CA harboring a beta -galactosidase reporter construct driven by p53-responsive elements (CA) was either treated with NaCl (-) or with 5 and 10 mM LiCl or irradiated with UV light for the indicated times. After cell lysis, beta -galactosidase activities were measured, and p21Cip expression was analyzed by immunoblotting. B, lithium-induced p53 activation and accumulation in BAEC. BAEC were transfected with 0.5 µg of p53-CA luciferase reporter construct (p53-CA-L) or with the luciferase reporter driven by the thymidine kinase minimal promoter (FOP). pRSV-beta -galactosidase vector was co-transfected for normalization of transfection efficiency. 24 h post-transfection, the cells were treated with 10 mM NaCl (C) or with 10 mM LiCl (Li) or were UV-irradiated (UV) for 6 and 12 h, and cell lysates were prepared. Luciferase activities were measured and normalized to beta -galactosidase activities. The results are mean ± S.D. of the normalized luciferase activity obtained for each condition in four independent experiments performed in duplicate. Expression of p53 and p21Cip was analyzed in parallel by immunoblotting.

The activation of p53 in response to lithium treatment was confirmed in BAEC by transient transfection of a luciferase reporter construct driven by p53-responsive elements (p53-CA-L) (51). After, 24 h of recovery, the transfected cells were treated with NaCl, LiCl, or UV irradiation for the indicated times, and luciferase activity of the cell extracts was determined. As shown in Fig. 7B, lithium treatment increased p53-dependent transcription of the luciferase reporter with a maximal effect of 2.3-fold increase at 9 h of treatment. BAEC irradiated with UV light displayed only a slight increase of 1.5-fold in the luciferase activity both at 6 and 9 h, although the accumulation of p21Cip protein was stronger in UV-irradiated cells as compared with lithium-treated cells (Fig. 7B). However, lithium was able to induce the accumulation of p53 protein after 12 h of treatment similarly to UV light irradiation, as detected by immunoblotting using the PAb240 monoclonal antibody (Fig. 7B). Therefore, these results showed that lithium induced both activation and accumulation of endogenous p53 in BAEC.

Lithium-induced p21Cip Expression Is Dependent on p53-- To determine if p53 mediated the induction of p21Cip expression by lithium, we investigated the effects of lithium in the mouse embryonic fibroblast MEFp53+/+, MEFp53+/-, and MEFp53-/-, cell lines, which are wild-type, heterozygous, and null for p53 alleles, respectively (45). MEFp53 cells were treated for 12 h with NaCl, LiCl, or UV light as a positive control, and cell extracts were prepared and analyzed for p21Cip expression by immunoblotting. Lithium, like UV irradiation, was able to induce p21Cip accumulation in MEF wild-type and heterozygous for the p53 alleles, but not in MEFp53 null cells (Fig. 8). These results suggest that lithium induced a p53-dependent pathway in various primary cells including BAEC.


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  		Fig. 8.   p53-mediated induction of p21Cip expression by lithium. Mouse embryonic fibroblasts wild-type for both alleles of p53 (Wt), deleted of one allele (Ht), or null for both alleles (N) were treated with either 5 mM NaCl (Control) or 5 mM LiCl (LiCl) or UV-irradiated (UV) for 12 h. Expression of p21Cip protein was analyzed in the cell lysates by immunoblotting.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this report, we have shown that in primary BAEC, lithium, an inhibitor of GSK-3beta and an activator of the Wnt-signaling pathway, induced stabilization and nuclear translocation of beta -catenin without inducing its transcriptional activity. Moreover, lithium induced in BAEC cell cycle arrest in the G2/M phase associated with the induction of p21Cip expression in a p53-dependent pathway. Lithium-induced cell cycle arrest was not accompanied by signs of apoptosis or loss of cell viability but rather with the appearance of a senescent-like phenotype. These effects are not restricted to BAEC but occur also in other primary cells: the mouse embryonic fibroblast and bovine vascular smooth muscle cells (data not shown). From these findings, we propose that beta -catenin stabilization and activation of p53 by lithium may account for its protective or survival effects reported in cells that were challenged by various stresses: radiation, ischemia, staurosporine, and glutamate excitotoxicity (39-42).

Although lithium induced beta -catenin stabilization in BAEC, it failed to induce beta -catenin transactivation functions as assessed with TCF-responsive luciferase reporter constructs (Fig. 1), whereas the epithelial cell line HEK293 demonstrated not only similar levels of beta -catenin stabilization and nuclear localization (Fig. 1) but also an increase of transcriptional activity as previously described (52). Similar observations have been made by others with the Jurkat T-cell line in comparison with the mouse epithelial cell line C57MG (55). We did not detect any activity of the beta -catenin-TCF complex in primary human umbilical vein EC (HUVEC) or in the immortalized microvascular EC cell line HMEC-1 (data not shown), although all of these EC expressed TCF4.2 In agreement with this absence of beta -catenin-TCF transcriptional activity in BAEC, we did not observe a significant increase in cyclin D1 expression, a known TCF target gene (16), in response to lithium (Fig. 5). Wright et al. (56) have shown that primary mouse brain microvascular EC in response to Wnt1 display beta -catenin-TCF transcriptional activity. Therefore, it is conceivable that additional modifications of beta -catenin and/or TCF factors that are not induced by lithium in EC must occur to get full activity. Indeed, both inactivation of GSK-3beta and activation of protein kinase C seem to be required for maximal activation of the beta -catenin-TCF complex in HEK293 cells (52).

Lithium induced cell cycle arrest in G2/M without affecting cell viability in BAEC (Figs. 2 and 3). In addition to inhibiting GSK-3beta , lithium is also known to inhibit the inositol monophosphatase 1, leading to inhibition of inositol phosphate dependent pathways via depletion of the cellular myoinositol pool (8). However, the addition of myoinositol did not prevent or rescue the inhibitory effects of lithium on cell cycle progression (Fig. 3A). Therefore, the lithium effects on cell cycle, like its developmental defects in Xenopus (8), do not correlate with the inhibition of inositol phosphate pathways. Recently, Smits et al. (34) have observed a similar G2/M cell cycle arrest in various transformed (P19 embryonal carcinoma, U2OS osteosarcoma, and SK-N-MC neuroepithelioma) or immortalized (NIH3T3) cell lines after lithium treatment. They have shown that the activity of the cyclin B-Cdc2 complex, required for the entry into mitosis, was impaired after lithium treatment due to sustained phosphorylation of Cdc2 on tyrosine residue 15 (34). Our results show that lithium is able to induce p21Cip expression, which is also a signal for cell cycle arrest in G2 (Fig. 5). Therefore, both mechanisms of G2 arrest are probably induced by lithium with induction of p21Cip responsible for the sustained arrest that we have observed (Table I and Fig. 3B) (57). Although inhibition of cell proliferation by beta -catenin signaling has not been described to date, the reported induction of cell apoptosis occurring after overexpression of beta -catenin (21-23) was probably preceded by cell cycle arrest. It will be very informative to determine whether stabilization of beta -catenin also induces a cell cycle arrest in G2.

Here, we have demonstrated that lithium is inducing stabilization and activation of p53 (Fig. 7) and that p21Cip induction is mediated by p53 (Fig. 8). Stabilization and activation of p53 is mediated mainly through specific phosphorylation induced by a variety of stimuli, which can be grouped in three classes: DNA-damaging agents such as UV light, agents that affect microtubules, and inappropriate spatio-temporal expression of factors involved in cell cycle regulation (58). We have observed a more spread morphology of BAEC after lithium treatment, indicating that changes in the organization of the cytoskeleton are occurring (Fig. 4). Changes in the microtubule network have been described in response to Wnt7A (28, 36) as well as in response to lithium and to dvl1, an upstream Wnt-signaling component mediating GSK3beta inactivation (59). Therefore, an increase in microtubule stability may be the signal for cell cycle arrest in the G2 phase, where disorganization of the microtubule must occur prior to formation of the mitotic spindle and cell rounding. Alternatively, beta -catenin may be the signal for cell cycle arrest. Indeed, Orford et al. (20) have shown that the nuclear localization of beta -catenin was cell cycle regulated in the epithelial Madin-Darby canine kidney cells with a peak during the S phase. Damalas et al. (60) have reported the accumulation of p53 in mouse fibroblasts NIH3T3 overexpressing a stable form of beta -catenin (S37A). It is thus possible that a sustained retention of beta -catenin in the nucleus during G2 or G2/M transition can signal for p53 induction and cell cycle arrest. On the other hand, stabilization of beta -catenin and p53 induced by lithium may occur in parallel through a similar mechanism. Further analyses are required to distinguish between these hypothesizes.

Some features of BAEC treated with lithium are associated with replicative senescence (46, 53): adoption of a flat and enlarged cell shape (Fig. 4), the appearance of SA-beta -galactosidase activity (Fig. 4), and reduced growth ability (Fig. 3) accompanied by the accumulation of p21Cip and p53 proteins (Figs. 5 and 7). Interestingly, changes in cell shape and appearance of multinucleated cells have been noted for renal mesenchymal cells constitutively expressing Wnt3A (61), which is similar to our observations with lithium-treated BAEC. Overexpression of p21Cip and p53 can cause premature senescence in low passage fibroblasts (62, 63). Therefore, further investigations are needed to determine whether lithium and abnormal activation of Wnt signaling may increase the rate of cell senescence.

Cell cycle-arrested or senescent cells display a reduced sensitivity to stress or DNA-damaging agents (53). The observed decrease in the apoptotic rate appears to be mediated by an activation of p53 and p53-dependent up-regulation of genes involved in DNA repair and cell survival. Once this survival state of the cell is established, p53 expression is dispensable (53). In addition, beta -catenin may also mediate cell survival. Indeed, previous reports have shown a correlation between decreases in beta -catenin levels and decreases in cell survival in response to anticancer drugs or stresses (21, 22, 64). The mechanisms of lithium action during bipolar therapy are not clearly understood, although recently a possible survival effect of lithium was proposed (1). Indeed, several studies have shown that lithium protects neuronal cells from undergoing apoptosis in response to a variety of insults, such as ischemia (40) or glutamate excitotoxicity (39). Activation of the phosphatidylinositol 3-kinase/AKT pathway (39) and inactivation of GSK-3beta (42) have been implicated in this protection, the effects of phosphatidylinositol 3-kinase/AKT pathway probably being mediated in part by inactivation of GSK-3beta (42). Down-regulation of p53 and Bax, a proapoptotic factor, in conjunction with an up-regulation of the antiapoptotic Bcl2 have also been proposed to mediate the protective effects of lithium in cerebellar granule cells (41). Such effects were only observed after a long term treatment of 7 days with lithium and, thus, are not in contradiction to our observations, since down-regulation of p53 is often observed after its activation (58). This down-regulation is due in part to an increase of MDM2 expression and of p53 targeting for degradation (58). Our results, in addition to strengthening the hypothesis of a cell survival effect of lithium in various cell types, suggest that stabilization of both p53 and beta -catenin may contribute to this effect.

Multinucleated and enlarged EC have been described in the aging vasculature but also in vascular lesions such as atherosclerosis and postangioplasty restenosis (65). Increases in p53 expression in EC as well as in smooth muscle cells and macrophages are associated with atherosclerotic lesions (32, 65). This increase appears to be part of the protective mechanisms that cells develop to limit the extent of proliferation in response to injury (33, 58). Indeed, in models of arterial injury, proliferation of vascular smooth muscle cells is inhibited by transfer of wild-type p53 with an absence of apoptosis (66), and conversely, abnormal proliferation of vascular smooth muscle cells is observed after transfer of p53 antisense oligonucleotide (67). Similar observations have been made in the double knockout mice p53-/- and ApoE-/-, where an acceleration of the atherosclerotic process is occurring as compared with ApoE-/- mice (68). This acceleration is mediated by an increase of cell proliferation and decrease of cell death. Therefore, lithium treatment, by inducing p53 and inhibiting cell proliferation in the absence of cell apoptosis, may also have a protective effect in vascular diseases. Although a direct role of the beta -catenin or GSK-3beta pathways has not been studied to date in the development of vascular diseases or in vascular cell survival, it is worth noting that the inactivation of GSK-3beta and stabilization of beta -catenin can occur in response to nitric oxide (69), a potent regulator of vascular functions. Therefore, lithium may be a good therapeutic agent in vascular diseases involving vascular cell proliferation, such as restenosis after coronary angioplasty.

    ACKNOWLEDGEMENTS

We thank Dr. Meredith Bond for critical reading of the manuscript. We thank Dr. Tyler Jacks for the gift of MEF-p53 cells, Dr. Micha Chernov for the gift of 12(1)CA cells and the p53-CA-driven reporter constructs, Dr. Hans Clevers for the TOPflash and FOPflash constructs, and Dr. Alexandru Almasan and Dr. Bendi Gong for the p21 promoter-driven luciferase constructs. We are especially thankful to Drs. Sara Carlson, Suddesh Aggrawal, and Guy Chisolm for the kind gifts of various antibodies and for p21Cip and p53 cDNAs. Amy Raber and Cathy Stanko, from the Cleveland Clinic Flow Cytometry Core, are acknowledged for assistance in performing FACScan analysis.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants HL29582 and HL34727 (to P. E. D.) and by American Cancer Society Institutional Research Grant IRG91-023-07 (to C. D. M.).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.

Dagger To whom correspondence should be addressed: Dept. of Molecular Cardiology, NB50, The Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-444-4673; Fax: 216-444-9263; E-mail: maoc@ccf.org.

Published, JBC Papers in Press, May 3, 2001, DOI 10.1074/jbc.M101188200

2 C. Mao, unpublished results.

    ABBREVIATIONS

The abbreviations used are: TCF, T-cell factor; EC, endothelial cell(s); BAEC, bovine aortic endothelial cell(s); MEF, mouse embryonic fibroblast; GSK-3beta , glycogen synthase kinase-3beta ; PBS, phosphate-buffered saline; SA-beta -galactosidase, senescent-associated beta -galactosidase; MOPS, 4-morpholinepropanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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