Overexpression of catalase delays G0/G1- to S-phase transition during cell cycle progression in mouse aortic endothelial cells

Abstract

Although it is understood that hydrogen peroxide (H(2)O(2)) promotes cellular proliferation, little is known about its role in endothelial cell cycle progression. To assess the regulatory role of endogenously produced H(2)O(2) in cell cycle progression, we studied the cell cycle progression in mouse aortic endothelial cells (MAECs) obtained from mice overexpressing a human catalase transgene (hCatTg), which destroys H(2)O(2). The hCatTg MAECs displayed a prolonged doubling time compared to wild-type controls (44.0 +/- 4.7 h versus 28.6 +/- 0.8 h, p<0.05), consistent with a diminished growth rate and H(2)O(2) release. Incubation with aminotriazole, a catalase inhibitor, prevented the observed diminished growth rate in hCatTg MAECs. Inhibition of catalase activity with aminotriazole abrogated catalase overexpression-induced antiproliferative action. Flow cytometry analysis indicated that the prolonged doubling time was principally due to an extended G(0)/G(1) phase in hCatTg MAECs compared to the wild-type cells (25.0 +/- 0.9 h versus 15.9 +/- 1.4 h, p< 0.05). The hCatTg MAECs also exhibited decreased activities of the cyclin-dependent kinase (Cdk) complexes responsible for G(0)/G(1)- to S-phase transition in the cell cycle, including the cyclin D-Cdk4 and cyclin E-Cdk2 complexes. Moreover, the reduction in cyclin-Cdk activities in hCatTg MAECs was accompanied by increased protein levels of two Cdk inhibitors, p21 and p27, which inhibit the Cdk activity required for the G(0)/G(1)- to S-phase transition. Knockdown of p21 and/or p27 attenuated the antiproliferative effect of catalase overexpression in MAECs. These results, together with the fact that catalase is an H(2)O(2) scavenger, suggest that endogenously produced H(2)O(2) mediates MAEC proliferation by fostering the transition from G(0)/G(1) to S phase.

Fig. 1. The effect of catalase overexpression on MAEC catalase activity andH₂O₂release

A. Catalsae activity. Wild-type and hCatTg MAECs were incubated in the culture medium in the presence or absence of 10% FBS and 10 μM aminotriazole (AT). Catalase activity was determined by measuring the decomposition of H₂O₂ using a spectrophotometric assay. Values represent the mean ± SEM of 5 separate experiments in which MAECs were pooled from 4 mice. *p<0.05 compared with wild-type cells. ^†p <0.05 compared with hCatTg cells without aminotriazole treatment. B. H₂O₂ release. Quiescent wild-type and hCatTg MAECs were seeded in medium supplemented with10% FBS for the time periods indicated. The release of H₂O₂ from MAECs was measured using an Amplex red hydrogen peroxide assay kit. Values represent the mean ± SEM of 6 separate experiments in which MAECs were pooled from 4 mice. *p<0.05 compared with wild-type cells without FBS incubation (0 h). ^†p <0.05 compared with wild-type cells at the same culture time period.

Fig. 2. The effect of catalase overexpression on MAEC proliferation

Quiescent. wild-type and hCatTg MAECs were cultured with 10% FBS for the time periods indicated in the presence or absence of 10 μM aminotriazole (AT). Cellular proliferation was determined based on ATP quantitation assays as described in the Materials and Methods. A. Cell growth curves. Cellular proliferation is expressed as the percentage increase from the number of cells initially seeded. B. Cell doubling times. The proliferation doubling time was calculated using an equation fit to the growth curve as described in the Materials and Methods. C. The effect of aminotriazole on catalase overexpression-induced antiproliferative action. Data represent the mean ± SEM of five separate experiments in which MAECs were pooled from four mice. *p<0.05 compared with wild-type cells under the same culture conditions.

Fig. 3. The effect of catalase overexpression on the distribution of MAECs in cell cycle phases

Quiescent wild-type and hCatTg MAECs were seeded in medium supplemented with 10% FBS at a density of 5×10⁵ cells per 100 mm dish. At the indicated time points, cells were trypsinized, fixed, stained with propidium iodide, and analyzed by flow cytometry. The right panel shows representative DNA histograms for MAECs. The left panel shows fraction (%) of cells distributed in various cell cycle phases at indicated timepoint. Data represent the mean ± SEM of five separate experiments in which MAECs were pooled from four mice. * P<0.05 as compared with wild-type cells under the same culture condition.

Fig. 4. The effect of catalase overexpression on the duration of cell cycle phases

Quiescent wild-type and hCatTg MAECs were seeded in medium supplemented with 10% FBS and harvested at 6 h intervals over a period of 48 h and analyzed using flow cytometry assays. We used the fraction of cells in each phase of the cell cycle, and the cell doubling time to calculate the duration of the cell cycle phases as described in the Materials and Methods. Data represent the mean ± SEM of five separate experiments in which MAECs were pooled from four mice. *p<0.05 compared with wild-type cells under the same culture condition.

Fig. 5. The effect of catalase overexpression on cyclin D-Cdk4 and cyclin E-Cdk2 kinase activities

Quiescent wild-type and hCatTg MAECs were seeded in medium containing 10% FBS and lysed at indicated time points. Activity of the CyclinD-Cdk4 and cyclinE-Cdk2 complexes were determined by in vitro kinase assays as described in the Material and Methods. The kinase activity is expressed as the radioactivity incorporated into the kinase substrates. Data represent the mean ± SEM of five separate experiments in which MAECs were pooled from four mice. *p<0.05 compared with 0 h of the same genotype cells. ^†p <0.05 compared with wild-type cells under the same culture conditions.

Fig. 6. The effect of catalase overexpression on the protein levels of cyclin D and Cdk4

Quiescent wild-type and hCatTg MAECs were seeded into dishes with medium containing 10% FBS and lysed at indicated time points. The levels of cyclin D1, cyclin D2, cyclin D3 andCdk4 were measured using western blot analysis and are expressed as the percentage of their immunoblot intensity relative to GADPH levels. The values represent mean ± SEM of five separate experiments. *p<0.05 compared with 0 h of the same genotype cells, ^†p <0.05 compared with wild-type cells at the same culture time points.

Fig. 7. The effect of catalase overexpression on the protein levels of cyclins and Cdks

Quiescent wild-type and hCatTg MAECs were seeded into dishes with medium containing 10% FBS and lysed at indicated time points. The levels of cyclin E and Cdk2 were measured using western blot analyses and are expressed as the percentage of their immunoblot intensity relative to GADPH levels. The values represent mean ± SEM of five separate experiments. *p<0.05 compared with 0 h of the same genotype cells, ^†p <0.05 compared with wild-type cells at the same culture time points.

Fig. 8. The effect of catalase overexpression on the protein levels of Cdk Inhibitors

Quiescent wild-type and hCatTg MAECs were seeded into dishes with medium containing 10% FBS and lysed at indicated time points. The protein levels of p27 and p21 were measured using western blot analyses and are expressed as the percentage of their immunoblot intensity relative to GAPDH levels. The values represent mean ± SEM of five separate experiments. *p<0.05 compared with 0 h of the same genotype cells, ^†p<0.05 compared with wild-type cells at the same culture time points.

Fig. 9. Knockdown of p21 and/or p27 increases cell proliferation in hCatTg MAECs

The hCatTg MAECs were transfected with p21 and/or p27 siRNA or control siRNA as described in the Materials and Methods. The transfected cells at confluence were made quiescent in serum-free DMEM for 12 h, and then incubated with 10% FBS for indicated time periods. The p21 and p27 levels were determined by western blot analysis, and expressed relative to GAPDH. Cell proliferation was determined by ATP quantitation assays, and expressed relative to the number of cells initially seeded. Values represent the mean ± SEM of five separate experiments in which MAECs were pooled from 4 mice * P< 0.05 compared with control siRNA-transfected cells, ^†p<0.05 compared with 0 h of the same genotype cells.