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Am J Physiol Lung Cell Mol Physiol 293: L702-L711, 2007. First published June 15, 2007; doi:10.1152/ajplung.00016.2007
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Predisposition to tetraploidy in pulmonary vascular smooth muscle cells derived from the Eker rats

Yu Gui, Gordon H. He, Michael P. Walsh, and Xi-Long Zheng

Smooth Muscle Research Group, Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada

Submitted 11 January 2007 ; accepted in final form 12 June 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Somatic mutations in the tuberous sclerosis complex-2 (TSC2) gene are associated with pulmonary lymphangioleiomyomatosis (LAM), a disorder characterized by benign lesions of smooth muscle and/or smooth muscle-like cells in the lung. However, the cellular mechanisms underlying LAM disease are largely unknown. Given that the TSC2 gene product tuberin is involved in the regulation of cell growth and proliferation, the present study was designed to investigate the potential roles of TSC2 in regulation of the cell cycle. We studied cell cycle profiles of pulmonary vascular smooth muscle cells (SMCs) derived from Eker rats (Tsc2+/EK), a genetic model carrying a germline insertional deletion in one copy of the Tsc2 gene, and the wild-type rats (Tsc2+/+), a noncarrier counterpart. We found that Tsc2+/EK, but not Tsc2+/+, SMCs displayed increases in cells with ≥4N DNA content (≥4N cells) and in the bromodeoxyuridine (BrdU) incorporation of ≥4N cells. Centrosome number was also increased in Tsc2+/EK SMCs, but the mitotic index was comparable between Tsc2+/+ and Tsc2+/EK SMCs. Furthermore, Tsc2+/EK SMCs showed elevated phosphorylation of p70S6K and increased expression of cell cycle regulatory proteins Cdk1, cyclin B, Cdk2, and cyclin E. Inhibition of the mammalian target of rapamycin (mTOR) pathway by rapamycin not only inhibited the phosphorylation of p70S6K and the expression of cell cycle regulatory proteins but also reduced accumulation of ≥4N cells and BrdU incorporation of >4N cells. Therefore, our results demonstrate that Tsc2+/EK SMCs are predisposed to undergo tetraploidization, involving activation of the mTOR pathway. These findings suggest an important role of Tsc2 in regulation of the cell cycle.

tuberous sclerosis complex-2; smooth muscle cell cycle; lymphangioleiomyomatosis

TUBEROUS SCLEROSIS COMPLEX (TSC) is an autosomal dominant disorder associated with benign hamartomas, which can occur in multiple organs, including brain, kidney, and lung. Genetic studies have demonstrated that somatic mutations in the TSC2 gene are linked to lymphangioleiomyomatosis (LAM) (4, 9), a disease characterized by benign lesions of smooth muscle and/or smooth muscle-like cells in the lung (42). LAM cells consist of phenotypically heterogeneous groups of smooth muscle cells (SMCs), mainly myofibroblast-like, spindle-shaped, and epithelioid cells (2). Although the origin of LAM cells is unclear, it has been hypothesized that pulmonary LAM cells may be derived from local vascular SMCs that undergo spontaneous mutations within the vessel walls or metastasize from a distant site, such as the kidney (8).

It has been suggested that TSC2, as a tumor suppressor gene, plays critical roles in regulation of cell growth and proliferation. Mice lacking the Tsc2 gene die in midgestation, and the Tsc2 heterozygotes develop cysts and slow-growing tumors in multiple organs (24, 28). Eker rats, which carry a germline insertional deletion of one copy of the Tsc2 gene, also develop tumors in a variety of tissues (23, 44, 45). Overexpression of Tsc2 in an Eker rat-derived kidney tumor cell line inhibits cell proliferation and suppresses tumorigenicity (21). The roles of TSC2 in regulation of cell growth and proliferation suggest that TSC2 product may be involved in regulation of the cell cycle. Indeed, in whole embryo cultures lacking Tsc2, there was sustained DNA synthesis in cardiomyocytes (32). Antisense inhibition of TSC2 expression induced the entry of quiescent fibroblasts into S phase and prevented cells from arrest at G1/0 in response to serum withdrawal (36). In addition, SMCs derived from lesions of LAM patients showed increased DNA synthesis (13, 14). These studies revealed that the TSC2 gene product might be involved in regulation of S-phase entry. Although it has been shown that loss of tuberin promotes S-phase entry of cells through affecting the stability of p27, a cyclin-dependent kinase inhibitor (37), the mechanisms underlying TSC2 regulation of the cell cycle are still largely unknown.

The TSC2 gene product, tuberin, has been recently linked to the mammalian target of rapamycin (mTOR) pathway, a signaling network involved in the regulation of cell growth and proliferation in response to growth factors and changes in cellular energy levels (25, 33, 34). Tuberin contains a putative GTPase-activating protein (GAP) domain at its COOH terminus, which can suppress the mTOR activity through inhibition of Rheb, a member of the Ras superfamily (11). Loss of tuberin results in activation of mTOR and phosphorylation of its downstream targets, p70S6K and 4E-BP1 (10, 26, 43). Importantly, upregulation of mTOR and activation of S6K have been observed in hamartomas from TSC patients and in SMCs derived from LAM lesions (6, 13). Inhibition of mTOR by rapamycin markedly inhibited phosphorylation of S6K and the elevated DNA synthesis in LAM-derived SMCs (13). These studies suggest that tuberin regulates the entry to S phase through suppression of the mTOR-S6K pathway.

Eukaryotic cells exhibit cell cycle variations. One common variation is endoreduplication cycling, in which cells increase their genomic DNA content without dividing and thus become polyploid (7). Endocycling cells utilize similar regulatory machinery as that of diploid cycling for regulation of G1-S transition, which requires the interaction of cyclin-dependent kinase-2 (Cdk2) and cyclin E. Interestingly, increased tetraploidy/polyploidy of SMCs has been observed in arterial walls of human and animals (1, 12) and found to be associated with aging and hypertension (31). SMC tetraploidization/polyploidization can be stimulated by angiotensin II in the presence of a mitotic inhibitor (19). The cellular events that promote SMC tetraploidization/polyploidization involve DNA duplication (endoreduplication) or aberrant mitosis (karyokinesis and/or cytokinesis failure) (16, 17, 29). Furthermore, in some TSC patients, the appearance of giant cells in TSC lesions is a major feature of brain tumors, raising the possibility that TSC is linked to the formation of polyploidy, since increased cell size appears to correlate with an increase in ploidy (39). However, studies in Drosophila show that mutations in TSC genes result in increase in size of cells but with normal ploidy (41). Therefore, whether increased polyploidy is associated with TSC hamartomas is unclear.

To investigate whether TSC2 is involved in regulation of the smooth muscle cell cycle, we took advantage of the fact that vascular SMCs have the potential to become tetraploid/polyploid and that Eker rats harbor Tsc2 genetic mutation. Cell cycle profiles of pulmonary vascular SMCs derived from Eker rats (Tsc2+/EK) and the wild-type counterparts (Tsc2+/+) were examined. In this article we report that Tsc2+/EK, but not Tsc2+/+, SMCs showed accumulation of cells with 4N DNA content (4N cells) and a fraction of >4N Tsc2+/EK cells continuing DNA synthesis. A potential role of the mTOR pathway in the increased 4N cells was also evaluated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. RPMI 1640 medium, fetal calf serum, trypsin-EDTA, and anti-bromodeoxyuridine (BrdU) monoclonal antibody were purchased from Invitrogen (Burlington, ON, Canada). Antibodies against cyclin B, cyclin E, Cdk2, and Cdk1 were obtained from BD Biosciences (Mississauga, ON, Canada). Rapamycin, PD 98059, anti-p27, anti-SM (smooth muscle) {alpha}-actin, and anti-phospho-histone H3 were obtained from Sigma (Oakville, ON, Canada). Anti-MAPK, anti-phospho-S6K, and anti-phospho-MAPK were obtained from Cell Signaling Technology (Beverly, MA).

Animal care and breeding. To establish an Eker rat colony, male Eker rats were transported from the Science Park-Research Division, University of Texas M. D. Anderson Cancer Center (Smithville, TX) to the Animal Resource Center at the University of Calgary and bred with female Long-Evans rats (Charles River, Senneville, QC, Canada), the background strain for the Eker mutation. All animals were cared for according to the recommendations of the Canadian Council on Animal Care. The animal study protocol was approved by the Animal Care and Use Committee at the University of Calgary. Male and female rats (2–3 mo of age and 250–300 g of body weight) were used in the current study.

Genotyping and Tsc2 allele analysis. Genotyping of the breeding colonies was performed using the polymerase chain reaction (PCR) method as described previously (20). Briefly, genomic DNA was extracted from ear tissue using a DNeasy kit (Qiagen, ON, Canada). PCR was carried out using a forward primer (5'-GAC TGG TAC TTC CTA GCA CCA T-3') and reverse primers (5'-AAA CTC CAC GCA TGC TCA GT-3' for the mutant and 5'-CTC GGC CTC CAA GTA CCA TCT-3' for the wild type) at 35 cycles of 95, 57, and 72°C for 30 s, respectively. The wild-type allele and the mutant allele were amplified as 237- and 183-bp products.

Cell culture and double thymidine block. After rats were killed by cervical dislocation, pulmonary arteries and arterioles were rapidly isolated. Pulmonary vascular SMCs derived from Eker and wild-type (noncarrier from the same colony) rats were obtained using the explantation method as described previously (5). Briefly, the isolated pulmonary arteries and arterioles at first order were dissected from adventitia tissue. After the vessels were opened, the endothelium was mechanically removed and the tissue cut into small pieces (1 mm3). The tissues were then plated in a primary cell culture dish. After culture for 3–5 days, cells grew out from the tissues. When cells reached subconfluence, the tissues were removed and the cells trypsinized for subculture. Cells were cultured in RPMI 1640 medium containing 10% fetal calf serum, and the medium was replaced every 2–3 days. The identity of SMCs was confirmed by immunostaining with antibodies against smooth muscle {alpha}-actin and h1-calponin. Allele analysis of cultured SMCs was also performed using the PCR method as described. After subculture for 3 days, cells were treated with and without different drugs for the periods of time indicated.

For double thymidine block, cells were incubated with 2 mM thymidine for 20 h, released into fresh medium for 8 h, incubated with 2 mM thymidine for 15 h, and then released into fresh medium. The time point right after release was set as 0 h. Cells were harvested from 0 up to 48 h, as indicated.

Laser scanning cytometer analysis. Cell cycle profiles and BrdU incorporation were analyzed as described previously (18). In brief, cultured cells were labeled with 10 mM BrdU for 60 min before harvesting and fixed in 80 and 100% ethanol. After incubation with 0.1% Triton X-100 and 4 N HCl, cells were immunostained with anti-BrdU antibody and a secondary antibody conjugated to Alexa Fluor-488. The nuclei were counterstained with propidium iodide (PI) in the presence of RNase. Cells were analyzed for their DNA contents and BrdU incorporation with a laser scanning cytometer (LSC).

Immunofluorescence study. Cells grown on coverslips were fixed with 4% paraformaldehyde for 20 min and permeabilized with 100% methanol overnight at –20°C. Cells were then blocked with 2% skim milk for 30 min. Mitotic index analysis was performed as described (17). Briefly, cells were stained with an antibody (1: 300 dilution) against phospho-histone H3 (serine-10), a mitotic marker, and a secondary antibody (1: 400 dilution) conjugated to Texas red. The nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). Cells were inspected with fluorescence microscopy and photographed with a charge-coupled device (CCD) camera. Mitotic cells were identified by the presence of condensed DNA and phospho-histone H3 (serine-10)-positive staining. The mitotic index was calculated as the percentage of mitotic cells vs. total cell count. A minimum of 300 cells was counted on each coverslip.

For observation of centrosomes, anti-{gamma} tubulin was used as a primary antibody. Cells were also double-stained with anti-{alpha}-tubulin or anti-PCNA (proliferating cell nuclear antigen) antibody, as indicated. Images were acquired with a laser confocal microscope (Leica DM RXA2) under a x40 or x100 oil-immersion lens and photographed with a Cooled Scientific CCD camera (Princeton Instruments, Trenton, NJ). Stacked images collected at 0.5-mm planes were analyzed using Imaris software.

Western blot detection. SMCs grown in 100-mm petri dishes were treated as indicated and lysed with 1 ml of ice-cold lysis buffer. After centrifugation (10,000 g for 15 min), the supernatants were collected for protein measurement using the Bradford assay. Equal amounts of protein samples (80 µg) were separated with 11% SDS-PAGE, followed by transfer to nitrocellulose membrane. Antibodies against tuberin, Cdk1, Cdk2, cyclin E, cyclin B, p27, phospho-S6K, MAPK, and phospho-MAPK were used as primary antibodies to detect the respective proteins. The secondary antibody was conjugated with horseradish peroxidase (1:3,000 dilution), and peroxidase activity was detected using an ECL detection kit (GE Healthcare, QC, Canada).

Cell sorting. Cultured Tsc2+/Ek SMCs were trypsinized and resuspended in 10 ml of PBS, followed by staining with Hoechst 3342 (10 µg/ml) for 10 min. The FACSVantage flow cytometer (Becton Dickinson), equipped with an automated cell deposition unit (ACDU) and an ultraviolet laser (wavelength = 351 nm), was used to sort cells. Based on the DNA content, cells were sorted into two groups of ≥4N and 2N cells. After sorting, cells were used for PCR and Western blot analysis as described earlier.

Statistical analysis. Results are means ± SE. Statistical comparisons were performed with Student's t-test for unpaired observations or one-way ANOVA for observations between multiple groups. A P value <0.05 was considered a significant difference.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
SMCs derived from pulmonary arterioles (arteries) of Eker rats exhibit accumulation of cells with ≥4N DNA content. To investigate whether the TSC2 gene affects the smooth muscle cell cycle, we prepared primary cultures of SMCs from pulmonary arteries and arterioles of Eker-carrier rats (Tsc2+/EK) and the noncarrier wild-type counterparts (Tsc2+/+) using the explantation method (5). The presence of the Eker insertion in one Tsc2 allele in cultured SMCs was confirmed using PCR analysis. PCR results showed that the wild-type allele displayed a 237-bp product and the Eker allele showed a 183-bp product (Fig. 1A), as detected previously (20).


Figure 1
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  		Fig. 1. Increased tetraploidy in pulmonary vascular smooth muscle cells (SMCs) derived from Eker rats. A: Tsc2 allele analysis. Pulmonary SMCs derived from Eker (Tsc2+/EK) and wild-type (Tsc2+/+) rats were used for DNA extraction and PCR analysis. The wild-type and Eker alleles amplified by PCR show 237- and 183-bp products, respectively. B: morphology of pulmonary vascular SMCs derived from wild-type (left) and Eker rats (right). Cultured SMCs were fixed with ethanol and stained with propidium iodide (PI), followed by inspection using fluorescence microscopy at x200 magnification. Arrows indicate Tsc2+/EK cells with enlarged nuclei. C and D: DNA content analyzed by laser scanning cytometer (LSC). Representative histograms of DNA content (detected as PI integral) vs. cell count in Tsc2+/+ (C) and Tsc2+/EK SMCs (D). Populations of cells with 2N, 4N, and >4N DNA content are summarized from 5 independent experiments.

 
During routine culture, we observed that Tsc2+/EK, but not Tsc2+/+, SMCs derived from female rats contained some cells with enlarged nuclei (Fig. 1B, arrows). In addition, cell cycle profiles detected by LSC showed that Tsc2+/EK SMCs exhibited a significant increase in cells with ≥4N DNA content (≥4N cells) (Fig. 1D) compared with Tsc2+/+ SMCs (Fig. 1C), suggesting the presence of tetraploid cells in SMCs derived from female Eker rats. The accumulation of ≥4N cells in Tsc2+/EK SMCs was observed from passages 2 to 8–10. Cells from passages 10–12 or subsequent subcultures did not display a significant increase in ≥4N cells, and cells with apparently normal nuclei became predominant. The increase in ≥4N cells was observed in six of nine female Eker rats but in none of 10 female wild-type rats. In addition, there was no significant difference in cell numbers between Tsc2+/EK and Tsc2+/+ cells during culture (data not shown). Among nine female Eker rats studied, two developed tumors in their kidneys at the same age (2–3 mo) as other Eker rats. SMCs derived from those two Eker rats did not show accumulation of ≥4N cells but displayed characteristics of senescence during passages 1–3 (data not shown). SMCs from one of nine female Eker rats had cell cycle profiles very similar to those from wild-type rats. In addition, SMCs derived from three of six male Eker rats demonstrated cell cycle profiles similar to those of SMCs derived from male wild-type rats. However, SMCs from the other three male Eker rats (Tsc+/EK) showed a slight increase in ≥4N cells (~20–25%) compared with those from the wild-type rats (Tsc+/+) (15–18%). Since ≥4N Tsc+/EK SMCs from the male rats were much less than those from the female ones (45–60%), we focused on the observations acquired from the majority of female Eker rats without further discussion of different Eker phenotypes observed.

To further characterize the accumulation of 4N cells, we monitored cell cycle progression through G2/M phase after release from G1/S arrest by double thymidine block (Fig. 2). In Tsc2+/+ SMCs, after double thymidine block, 84% of cells were synchronized at G1/S phase (2N DNA content), only 14% of cells exhibited 4N DNA content (4N cells), and 2% of cells exhibited >4N DNA content. Six hours after release from thymidine block, Tsc2+/+ SMCs entering into G2/M phase reached a maximal level (48 ± 3%, n = 4) and gradually declined toward the basal level at 48 h after release. No significant difference between >4N cells was detected in Tsc2+/+ SMCs at different times after release (n = 4, P > 0.05). However, 4N cells in Tsc2+/EK groups after double thymidine block were as high as 30%, concurrent with the presence of 13% of >4N cells. 4N cells in Tsc2+/EK also reached the maximal levels (40 ± 2%) at 6 h after release, although accumulation of 4N cells from Tsc2+/EK groups was less compared with that (48 ± 3%) in Tsc2+/+ cells (n = 4, P < 0.05). Note that in Tsc2+/EK SMCs, there were still 32% of 4N cells after release for 48 h. The number of >4N cells reached a maximal level (25%) at 12 h after release and declined to 15% at 48 h after release. These results demonstrate three findings. 1) Tsc2+/EK SMCs contained a sustained portion of 4N and >4N cells, which could not be synchronized by double thymidine block. The number of 4N cells in Tsc2+/EK SMCs was still high at 48 h after release from the block, suggesting a 4N phase arrest. 2) There was no obvious delay in cell cycle progression through 2N phases into 4N phases between Tsc2+/EK and Tsc2+/+ SMCs, since 4N cells reached the maximal levels at 6 h after release in both groups. 3) The numbers of >4N cells increased from 13% (before release) to 25% (at 12 h) and then declined to 15% (at 48 h) after release, suggesting that Tsc2+/EK SMCs continued the cell cycle with 4N DNA content.


Figure 2
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  		Fig. 2. Cell cycle profiles of Tsc2+/EK SMCs after synchronization. Tsc2+/+ and Tsc2+/EK SMCs were synchronized by double-thymidine treatment as described in MATERIALS AND METHODS. After release from double-thymidine block at the times indicated, cells were fixed and stained with PI in the presence of RNase, followed by LSC analysis to detect the cell cycle distribution based on their DNA contents. The percentages of 4N and >4N cells are summarized from 3 independent experiments.

 
4N cells could be in G2 phase, mitotic phase, or tetraploid G1/0. Since cells with enlarged nuclei did not display condensed DNA, it was suggested that 4N cells were not in mitotic phase. To confirm this, cells were stained with an antibody against phospho-histone H3 (serine-10), a mitotic marker, with the nuclei counterstained with DAPI. The mitotic index analysis revealed that both Tsc2+/+ and Tsc2+/EK SMCs had comparable numbers of mitotic cells (n = 4, P > 0.05) (Fig. 3), supporting the conclusion that 4N cells were not the cells arrested in mitotic phase. Thus Tsc2+/EK SMCs may arrest in G2 phase or tetraploid G1/0 phases. Since cells at G2 phase should have two centrosomes, whereas cells at tetraploid G1/0 may have multiple centrosomes (3), we detected centrosome numbers in Tsc2+/EK SMCs. Immunostaining for {gamma}-tubulin, a centrosome component, indicated that 34% of Tsc2+/EK SMCs demonstrated increased numbers of centrosomes (Fig. 4). However, the majority of Tsc2+/+ SMCs contain two or fewer centrosomes. These results suggested that 4N cells could be tetraploid G1/0 cells and Tsc2+/EK SMCs were predisposed to undergo tetraploidization.


Figure 3
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  		Fig. 3. Analysis of the mitotic index in Tsc2+/+ and Tsc2+/EK SMCs. Cells cultured in RPMI 1640 medium with 10% FBS for 3 days were fixed with methanol and stained with anti-phospho-histone H3. The nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). Mitotic cells were identified by their condensed DNA and the positive staining for phospho-histone H3. The mitotic index was expressed as a percentage of mitotic cells vs. total cell count.

 

Figure 4
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  		Fig. 4. Centrosome detection in Tsc2+/+ and Tsc2+/EK SMCs. A: cells were fixed with methanol and stained with {alpha}-tubulin (green) and {gamma}-tubulin (red). The nuclei were stained with DAPI (blue). Fluorescence images were acquired by confocal microscopy using a x40 objective. Arrows indicate centrosomes. B: centrosomes were scored from 300 cells on each coverslip. Data were summarized from 3 experiments. *Significant difference compared with Tsc2+/+ groups.

 
Tetraploid Tsc2+/EK cells continue synthesizing DNA. To determine whether tetraploid Tsc2+/EK SMCs indeed entered the next cell cycle, we detected DNA synthesis in 4N cells by labeling cells with BrdU for 60 min under unsynchronized conditions. LSC analysis revealed that the total BrdU incorporation rates were not significantly different between Tsc2+/EK and Tsc2+/+ SMCs (Fig. 5, A and B). However, there was a marked increase in BrdU-positive cells with >4N DNA content in Tsc2+/EK groups compared with Tsc2+/+ SMCs (7.5% vs. 0.5%, n = 5, P < 0.01). The BrdU-positive >4N cells accounted for almost 50% of total BrdU-positive cells in Tsc2+/EK SMCs. The insets in Fig. 5, A and B, represent corresponding BrdU-positive cells with DNA content ≤4N and >4N detected by the relocation feature of LSC. These results suggested that 4N Tsc2+/EK SMCs continued to reduplicate their DNA. To seek more evidence, we further examined whether S-phase cells with enlarged nucleus contained multiple centrosomes by coimmunostaining cells with antibodies against PCNA and {gamma}-tubulin. The results showed that in Tsc2+/+ SMCs, 12 of 12 PCNA-positive cells examined by confocal microscopy contained one centrosome with one pair of centrioles (Fig. 5C). However, in Tsc2+/EK SMCs, 8 of 12 PCNA-positive cells showed one or two centrosomes, each of which contained four pairs of centrioles (Fig. 5D). These results further supported the conclusion that tetraploid/polyploid Tsc2+/EK SMCs continued reduplicating their DNA.


Figure 5
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  		Fig. 5. Active DNA synthesis in tetraploid or polyploid Tsc2+/EK cells. A and B: bromodeoxyuridine (BrdU) incorporation of Tsc2+/+ and Tsc2+/EK SMCs detected by LSC. The left and right upper quadrants define BrdU-positive cells with ≤4N and >4N DNA content; insets show nuclear images of BrdU-positive cells with ≤4N and >4N DNA content, respectively, detected by the relocation features of LSC. The numbers of total BrdU-positive and BrdU-positive >4N cells are summarized from 5 independent experiments. C and D: confocal images (taken under x100 oil objective) of Tsc2+/+ (C) and Tsc2+/EK SMCs (D) stained with PCNA (green), {gamma}-tubulin (red), and DAPI (blue). Images in D show the centrosomes collected at different stacks of 1 Tsc2+/EK cell.

 
Upregulation of Cdk1, Cdk2, cyclin B, and cyclin E in Tsc2+/EK SMCs. To further investigate the roles of TSC2 product in regulation of the cell cycle, we detected expression of cell cycle regulatory proteins. Western blot analysis showed that the expression of Cdk1, cyclin B, Cdk2 and cyclin E was increased in Tsc2+/EK SMCs compared with Tsc2+/+ SMCs (Fig. 6A). However, the levels of Cdk inhibitors, p27 and p21, were not significantly different between Tsc2+/+ and Tsc2+/EK SMCs (Fig. 6A and not shown). To determine whether ≥4N cells contribute to the increased expression of cell cycle regulatory proteins in Tsc+/EK SMCs, cells were sorted into 2N and ≥4N groups based on their DNA contents. As anticipated, the expression levels of cyclin B and cyclin E in ≥4N cells were much higher than those in 2N cells (Fig. 6A). Sorted ≥4N cells were also used to detect Tsc2 alleles by PCR analysis as described in MATERIALS AND METHODS. We found that ≥4N cells had the same two-band pattern as that of the nonsorting Tsc+/EK SMCs as shown in Fig. 1A (data not shown). Next, we examined the cellular distribution of cyclin B by immunostaining. We observed that in both Tsc2+/+ and Tsc2+/EK SMCs, nuclear signals for cyclin B were strong in mitotic cells and very weak in some interphase cells (Fig. 6B, circles). In Tsc2+/EK SMCs, however, cells with enlarged nuclei contained signals for cyclin B in both the nucleus and cytoplasm (Fig. 6B, arrow).


Figure 6
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  		Fig. 6. Upregulation of Cdk1, Cdk2, cyclin E, and cyclin B expression in Tsc2+/EK SMCs. A: representative results of Western blot detection of cell cycle regulatory proteins. Cells either cultured in medium with 10% FBS or sorted by fluorescence-activated cell sorting (FACS) flow cytometry based on their DNA contents were lysed for protein extraction. Protein (80 µg) from each sample were subjected to 11% SDS-PAGE and blotted with antibodies against cyclin B, Cdk1, Cdk2, cyclin E, and p27, respectively. Smooth muscle {alpha}-actin was used as a loading control. B: cellular distribution of cyclin B. Cells were fixed and immunostained with anti-cyclin B (green) and DAPI (blue). Arrows indicate the cytoplasmic and nuclear distribution of cyclin B in cells with enlarged nuclei. Circles indicate negative staining for cyclin B in interphase cells.

 
Tetraploidization in Tsc2+/EK SMCs involves the mTOR-S6K but not the MAPK pathway. Note that loss of the TSC2 gene results in activation of the mTOR pathway. Hyperphosphorylation of S6K was observed in LAM-derived SMCs (13). In addition, a high level of MAPK activation was found in giant cell astrocytomas from TS patients (15). To investigate whether the mTOR-S6K and/or MAPK pathways were involved in tetraploidization of Tsc2+/EK SMCs, we first examined the effects of rapamycin (an mTOR inhibitor) and PD 98059 (a MAPK kinase inhibitor) on BrdU incorporation rate and the cell cycle profiles. Representative histograms and summarized data (Fig. 7A) show that in Tsc2+/EK SMCs, treatment with rapamycin (50 nM), but not PD 98059 (20 µM), inhibited BrdU incorporation rate, especially in the BrdU-positive >4N cells. Both 4N and >4N cells in Tsc2+/EK SMCs were reduced by treatment with rapamycin compared with the untreated control (28 ± 3.3 and 5 ± 1.9% vs. 38 ± 4.2 and 13 ± 2.3%, respectively) (P < 0.01, n = 5). In contrast, in Tsc2+/+ SMCs, treatment with rapamycin only slightly reduced the cell numbers in S phase (P > 0.05, n = 5). These results suggested that the mTOR pathway, but not the MAPK pathway, was involved in increased tetraploidy in Tsc2+/EK SMCs. To further investigate the involvement of the mTOR-S6K and the MAPK pathways, we analyzed phosphorylation of S6K and MAPK in Tsc2+/+ and Tsc2+/EK SMCs in the absence and presence of either rapamycin or PD 98059. Western blot analysis showed that phosphorylated MAPK and S6K were upregulated in Tsc2+/EK SMCs compared with Tsc2+/+ SMCs. However, the expression of total MAPK or S6K was unchanged. The presence of rapamycin, which inhibited the accumulation of ≥4N cells and BrdU-positive >4N cells in Tsc2+/EK SMCs (Fig. 7A), markedly reduced phosphorylation of S6K (Fig. 7B), supporting a role for the mTOR pathway in tetraploidization. The presence of PD 98059 inhibited the phosphorylation of MAPK (Fig. 7B) but did not affect the accumulation of ≥4N cells or BrdU-positive >4N cells (Fig. 7A). Consistently, in Tsc2+/EK SMCs, treatment with rapamycin, but not PD 98059, reduced the increased centrosome numbers (data not shown). To determine whether activation of the mTOR-S6K pathway observed in tetraploidization of Tsc2+/EK SMCs is involved in regulation of cell cycle regulatory proteins, we examined the expression of cyclins and Cdks in the presence of rapamycin. Western blot analysis showed that treatment with rapamycin, which inhibited both phosphorylation of S6K and tetraploidization of Tsc2+/EK SMCs, prevented the increases in expression of cyclins B and E and Cdks 1 and 2 in Tsc2+/EK SMCs (Fig. 7C).


Figure 7
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  		Fig. 7. Tetraploidization and increased DNA synthesis in Tsc2+/EK SMCs involve the mTOR-S6K, but not the MAPK, pathway. A: cell cycle profiles of Tsc2+/+ and Tsc2+/EK SMCs in the presence of rapamycin (Rapa) and PD 98059. Cultured SMCs, incubated with or without either rapamycin (50 nM) or PD 98059 (20 µM) for 2 days, were fixed and processed for cell cycle analysis by LSC. The number of cells with 2N, 4N, and >4N DNA content and S-phase cells are summarized from 5 experiments. B: Western blot analysis of phospho-S6K, phospho-MAPK, S6K, and total MAPK from Tsc2+/+ and Tsc2+/EK SMCs. Cultured SMCs, treated with or without either rapamycin or PD 98059 for 2 days, were lysed for protein extraction. Protein samples (80 µg of protein from each lysate) were used for Western blot detection with anti-phospho-S6K, anti-phospho-MAPK, and anti-MAPK antibodies, respectively. C: Western blot analysis of cyclin B, cyclin E, Cdk1, and Cdk2 in the same cell lysates as in B. Data are representative of 3 independent experiments with similar results.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study reports that cultured SMCs derived from pulmonary arterioles and arteries of Eker rats (Tsc2+/EK), but not the wild-type counterparts (Tsc2+/+), exhibited a spontaneous increase in cells with 4N DNA content. The 4N cells did not contain condensed DNA but had increased centrosomes, suggesting that the cells were at tetraploid stages. In addition, the appearance of BrdU-positive >4N cells and the increase of >4N cells at 12 h that declined at 24 h after release from double thymidine block indicate the cycling of tetraploid cells. Thus Tsc2+/EK pulmonary vascular SMCs during primary cell culture are predisposed to tetraploidy/polyploidy, and these tetraploid cells can reenter the cell cycle with 4N DNA contents.

TSC disease is characterized by the occurrence of benign hamartomas in different organs. In the brain, TSC lesions contain characteristic giant cells (22, 35). In the lung, TSC2 mutation-associated LAM disease is characterized by deregulated proliferation of smooth muscle and/or smooth muscle-like cells. However, whether multinucleated SMCs exist in LAM lesions is still unclear. Our observation that predisposition to tetraploidy in pulmonary SMCs derived from Eker rats has revealed the complexity of proliferation properties of Tsc2+/EK pulmonary SMCs and their relevance to Tsc2 gene functions. First, polyploidy/aneuploidy is well known to cause genomic instability, leading to deregulated cell proliferation and development of cancer (38). The observation that Tsc2+/EK pulmonary SMCs underwent polyploidization raised the possibility that tetraploidy/polyploidy of local pulmonary vascular SMCs could contribute to the formation of LAM cells. In addition, our observation that tetraploidy/polyploidy was more significant in female compared with male Eker rats echoes the fact that LAM disease occurs exclusively in women. However, more detailed investigations are required to confirm whether the observations from in vitro studies reflect the situation in vivo.

Genetic studies indicate that TSC diseases are associated with loss of heterozygosity (LOH) and inactive mutations in one of two TSC genes. In the present study, Tsc2 genes in Tsc2+/EK SMCs and sorted 4N Tsc2+/EK cells, as detected by PCR, showed one normal allele and one Eker mutant allele, suggesting that tetraploidization/polyploidization of Tsc2+/EK SMCs was unlikely to result from LOH. Because of the limitations of the approach used, however, whether any Tsc2 mutations other than Eker mutation or haploinsufficiency lead to tetraploidization/polyploidization in Tsc2+/EK SMCs remains to be determined.

Vascular SMCs have the potential to undergo tetraploidization/polyploidization under certain circumstances, as is evident from the increased polyploidy/aneuploidy found in arterial walls of animals and human (1, 12) and associated with aging and hypertension (31). In addition, SMC polyploidization can be stimulated by angiotensin II in the presence of a mitotic inhibitor (19). The cellular events resulting in mitotic inhibitor-induced vascular SMC polyploidization involve induction of aberrant mitosis and endoreduplication (16, 17). In Tsc2+/EK SMCs, we observed increased tetraploidy/polyploidy without evidence of abnormal mitosis, and a fraction of tetraploid Tsc2+/EK cells continued DNA synthesis. These results suggest that tetraploid SMCs reenter the next S phase and undergo cycling. Consistent with the view of tetraploid SMC cycling, it was found that tetraploid SMCs derived from the spontaneous hypertensive rats proliferated in response to growth factors (30).

The molecular mechanisms underlying SMC tetraploidization/polyploidization are unclear. It has been suggested that Akt is involved in promotion of SMC polyploidization, since overexpression of Akt promotes mitotic inhibitor-triggered polyploidization of SMCs (19). Interestingly, it was reported that the mTOR-S6K pathway downstream of Akt was required for endocycling in Drosophila (46). In the present study we found that the mTOR pathway is involved in tetraploidization/polyploidization in Tsc2+/EK SMCs. This conclusion is supported by two lines of evidence: 1) Tsc2+/EK cells displayed increased phosphorylation of S6K, suggesting activation of the mTOR pathway in these cells; and 2) rapamycin, an inhibitor of mTOR, inhibited not only phosphorylation of S6K but also the increases in tetraploid/polyploid cells and BrdU-positive >4N cells. Our data are consistent with the findings that upregulation of mTOR and activation of S6K were present in TSC hamartomas from patients and in SMCs derived from LAM tissues (6, 13) and that inhibition of mTOR by rapamycin markedly inhibited phosphorylation of S6K and the increased DNA synthesis in LAM-derived SMCs (13).

The roles of TSC2 in regulation of the cell cycle are largely unknown. Previous studies suggested that TSC2 may be involved in the regulation of S-phase entry (13, 36, 37). The molecular mechanisms underlying TSC2-mediated S-phase entry involve upregulation of G1 cyclin, cyclin D (36), and a decrease in p27 stability (37). Our results showed that the levels of Cdk2 and cyclin E were increased in Tsc2+/EK SMCs compared with Tsc2+/+ SMCs, consistent with the previous finding that loss of TSC2 caused upregulation of G1 cyclins (36). Importantly, our results also showed that the expression of cyclin B was increased in Tsc2+/EK SMCs. Cyclin B was observed in both the nucleus and cytoplasm of Tsc2+/EK SMCs with enlarged nuclei. Furthermore, inhibition of the mTOR-S6K pathway by rapamycin inhibited the accumulation of 4N cells and BrdU-positive >4N cells and also reduced the increased expression of Cdk1, Cdk2, cyclin E, and cyclin B. Given that entry into mitosis requires translocation of cyclin B to the nucleus (40) and that cyclin B has a potential role in the promotion of S-phase entry (27), it is reasonable to assume that the increase in expression of cyclin B in 4N Tsc2+/EK cells contributes not only to accumulation of 4N cells but also continuous DNA synthesis without completion of mitosis in 4N cells. Therefore, tetraploidization/polyploidization of Tsc2+/EK SMCs could result from upregulation of cell cycle regulatory proteins through activation of the mTOR-S6K pathway. To our knowledge, this is the first report that tetraploidization/polyploidization occurs in Tsc2+/EK SMCs. Normal SMCs have the potential to undergo tetraploidization/polyploidization in response to various stimuli (1, 12, 19). Tetraploidization/polyploidization of Tsc2+/EK SMCs strongly suggests a stimulatory role of the Eker mutation in cell cycle progression into S phase and upregulation of cell cycle regulatory proteins. The effects of rapamycin have provided additional support for this concept, but more studies are required to elucidate the detailed mechanism.

Our data demonstrate that pulmonary vascular SMCs derived from the Eker rat are predisposed to tetraploidy/polyploidy, which involves activation of the mTOR-S6K pathway and upregulation of cell cycle regulatory proteins. This work sheds light on the roles of TSC2 in regulation of the cell cycle.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by a grant from Tuberous Sclerosis Alliance and TSC Canada (to X.-L. Zheng), an equipment grant from the Canada Foundation for Innovation (to M. P. Walsh), and a grant from the Alberta Lung Association (to X.-L. Zheng). X.-L. Zheng and Y. Gui are recipients of a New Investigator Award and a Postdoctoral Fellowship Award, respectively, from the Heart & Stroke Foundation of Canada. M. P. Walsh is the recipient of a Canada Research Chair (Tier 1) in Vascular Smooth Muscle Research and is an Alberta Heritage Foundation for Medical Research Senior Scientist.


   ACKNOWLEDGMENTS
 
We are very grateful to Dr. Cheryl L. Walker for providing the Eker breeders and critical reading of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: X.-L. Zheng, Dept. of Biochemistry & Molecular Biology, Faculty of Medicine, Univ. of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1 (e-mail: xlzheng{at}ucalgary.ca)

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.


   REFERENCES
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 MATERIALS AND METHODS
 RESULTS
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 REFERENCES
 

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