Endothelial cells isolated from caveolin-2 knockout mice display higher proliferation rate and cell cycle progression relative to their wild-type counterparts - PubMed (original) (raw)

Endothelial cells isolated from caveolin-2 knockout mice display higher proliferation rate and cell cycle progression relative to their wild-type counterparts

Leike Xie et al. Am J Physiol Cell Physiol. 2010 Mar.

Abstract

The goal of this study was to determine whether caveolin-2 (Cav-2) is capable of controlling endothelial cell (EC) proliferation in vitro. To realize this goal, we have directly compared proliferation rates and cell cycle-associated signaling proteins between lung ECs isolated from wild-type (WT) and Cav-2 knockout (KO) mice. Using three independent proliferation assays, we have determined that Cav-2 KO ECs proliferate by ca. 2-fold faster than their WT counterparts. Cell cycle analysis by flow cytometry of propidium iodide-stained cells showed a relatively higher percentage of Cav-2 KO ECs in S and G(2)/M and lower percentage in G(o)/G(1) phases of cell cycle relative to their WT counterparts. Furthermore, an over 2-fold increase in the percentage of S phase-associated Cav-2 KO relative to WT ECs was independently determined with bromodeoxyuridine incorporation assay. Mechanistically, the increase in proliferation/cell cycle progression of Cav-2 KO ECs correlated well with elevated expression levels of predominantly S phase- and G(2)/M phase-associated cyclin A and B1, respectively. Further mechanistic analysis of molecular events controlling cell cycle progression revealed increased level of hyperphosphorylated (inactive) form of G(1) to S phase transition inhibitor, the retinoblastoma protein in hyperproliferating Cav-2 KO ECs. Conversely, the expression level of the two cyclin-dependent kinase inhibitors p16(INK4) and p27(Kip1) was reduced in Cav-2 KO ECs. Finally, increased phosphorylation (activation) of proproliferative extracellular signal-regulated kinase 1/2 was observed in hyperproliferating Cav-2 KO ECs. Overall, our data suggest that Cav-2 negatively regulates lung EC proliferation and cell cycle progression.

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Figures

Fig. 1.

Fig. 1.

Isolation and initial characterization of mouse lung endothelial cells (MLECs). A: Western blot characterization of the purity of isolated wild-type (WT) and caveolin-2 (Cav-2) knockout (KO) MLECs relative to “lung cells” remaining after first immunoisolation with anti-platelet endothelial cell adhesion molecule-1 (PECAM-1) antibody. Note that, unlike WT, Cav-2 KO MLECs and lung cells are completely devoid of Cav-2. Both WT and Cav-2 KO MLEC protein lysates are positive for endothelial cell (EC)-specific marker proteins, i.e., Flk-1, PECAM-1, VE-cadherin, and endothelial nitric oxide synthase (eNOS), but negative for the mural cell marker protein α-actin. Conversely, protein lysates from lung cells are negative for EC marker proteins but positive for α-actin. B: immunofluorescent microscopy images of MLECs colabeled with 4′-6-diamidino-2-phenylindole (DAPI; blue nuclear staining) and with antibodies against EC marker proteins: VE-cadherin (green plasma membrane staining) or von Willebrand factor (vWF; green perinuclear staining). Note that all WT and Cav-2 KO cells are VE-cadherin or vWF positive. Hsp-90, heat shock protein 90.

Fig. 2.

Fig. 2.

Higher proliferation rate in Cav-2 KO relative to WT MLECs. A: direct determination of cell proliferation rate by cell count using hemocytometer. MLECs were plated at 4 × 104 cells per 60-mm dish, and cell numbers were determined at the indicated time points by cell count and are expressed as means ± SD (n = 9). Values are from one representative out of a total of 3 experiments. B: indirect determination of cell proliferation rate by colorimetric assessment of MTT to formazan conversion. MLECs were plated at 2.5 × 103 cells per well of 96-well plate, and optical density (OD) was measured at the indicated time points using a plate reader with a test wavelength of 550 nm and a reference wavelength of 690 nm to obtain a sample signal (550–690 nm). Data are expressed as means ± SD (n = 3). Values are from one representative out of a total of 4 experiments. C: indirect determination of cellular proliferation rate by colorimetric assessment of cellular DNA staining with methylene blue. MLECs were plated at 5 × 103 per well in a 96-well plate, and OD was measured at the indicated time points using a plate reader with a test wavelength of 655 nm. Data are expressed as means ± SD (n = 3). Values are from one representative out of a total of 4 experiments.

Fig. 3.

Fig. 3.

Increased association in hyperproliferating Cav-2 KO MLECs with S and G2/M phases and the concurrent decreased association with G0/G1 phases of cell cycle determined by flow cytometry. WT and Cav-2 KO MLECs were plated at 3 × 105 per 150-mm dish, and 2 days later, harvested with trypsin, fixed in ethanol, stained with propidium iodide, and analyzed by flow cytometry using fluorescent channel 2 (FL2-area). Representative cell cycle profiles from one representative out of a total of 3 experiments are shown for WT (A) and Cav-2 KO MLECs (B). Quantitative assessment of the percentage of cellular population associated with each phase of the cell cycle is illustrated in C.

Fig. 4.

Fig. 4.

Enhanced S phase association in hyperproliferating Cav-2 KO MLECs determined by BrdU incorporation assay. A and B: WT (A) and Cav-2 KO (B) MLECs were plated at 5 × 103 per chamber of an 8-chamber glass slide precoated with 0.2% gelatin, and 3 days later, pulsed with 5-bromo-2-deoxyuridine (BrdU) for 2 h. Fixing and immunofluorescence staining with fluorescein isothiocyanate-conjugated anti-BrdU antibody (green) and DAPI (blue) was then performed. Note the considerably higher number of BrdU-positive (green) cells in Cav-2 KO (B) relative to WT MLECs (A). C: % BrdU-positive cells was calculated and data are expressed as means ± SD (n = 5). Values are from one representative out of a total of 3 experiments.

Fig. 5.

Fig. 5.

Elevated expression levels of S and G2/M phase associated cyclins A and B1 in hyperproliferating Cav-2 KO MLECs relative to their WT counterparts determined by immunoblotting. A: WT and Cav-2 MLECs were plated at 3 × 105 per 150-mm dish, and 2 days later, cells were lysed and SDS-PAGE-resolved protein lysates were immunoblotted with the indicated cyclin-specific antibodies. B_–_E: mean densitometric ratios ± SD (n = 3) of cyclin proteins expression levels to protein loading control Hsp-90. Note a visible increase in the expression levels of cyclins A and B1 but not D1 and E in Cav-2 KO relative to WT MLECs.

Fig. 6.

Fig. 6.

Increased levels of phosphorylated/inactive form of the Rb protein with concurrent decrease in the expression levels of cyclin-dependent kinase (cdk) inhibitors p27Kip1 and p16INK4 in hyperproliferating Cav-2 KO relative to WT MLECs determined by immunoblotting. A: WT and Cav-2 MLECs were plated at 3 × 105 per 150-mm dish, and 2 days later, lysed and immunoblotted with the indicated antibodies. B: mean densitometric ratios ± SD (n = 3) of phosphorylated (inactive) Rb levels to protein loading control Hsp-90. C and D: mean densitometric ratios ± SD (n = 3) of indicated cdk inhibitors p16INK4 (p16) and p27Kip1 (p27) expression levels to protein loading control Hsp-90.

Fig. 7.

Fig. 7.

Augmented activation/phosphorylation state of ERK1/2 in hyperproliferating Cav-2 KO relative to WT MLECs determined by immunoblotting. A: WT and Cav-2 KO MLECs were plated at 3 × 105 per 150-mm dish, and 2 days later, were lysed and immunoblotted with the indicated antibodies. B: mean densitometric ratios ± SD (n = 3) of phosphorylated ERK1/2 (phospho-ERK1/2) to total expression level (ERK1/2).

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