Cyclin A transcriptional suppression is the major mechanism mediating homocysteine-induced endothelial cell growth inhibition

Abstract

Previously, it was reported that homocysteine (Hcy) specifically inhibits the growth of endothelial cells (ECs), suppresses Ras/mitogen-activated protein (MAP) signaling, and arrests cell growth at the G(1)/S transition of the cell cycle. The present study investigated the molecular mechanisms underlying this cell-cycle effect. Results showed that clinically relevant concentrations (50 microM) of Hcy significantly inhibited the expression of cyclin A messenger RNA (mRNA) in ECs in a dose- and time-dependent manner. G(1)/S-associated molecules that might account for this block were not changed, because Hcy did not affect mRNA and protein expression of cyclin D1 and cyclin E. Cyclin D1- and E-associated kinase activities were unchanged. In contrast, cyclin A-associated kinase activity and CDK2 kinase activity were markedly suppressed. Nuclear run-on assay demonstrated that Hcy decreased the transcription rate of the cyclin A gene but had no effect on the half-life of cyclin A mRNA. In transient transfection experiments, Hcy significantly inhibited cyclin A promoter activity in endothelial cells, but not in vascular smooth muscle cells. Finally, adenovirus-transduced cyclin A expression restored EC growth inhibition and overcame the S phase block imposed by Hcy. Taken together, these findings indicate that cyclin A is a critical functional target of Hcy-mediated EC growth inhibition.

Figure 1. The inhibitory effect of Hcy on DNA synthesis is reversible in ECs

HUVECs were exposed to DL-Hcy for 24 hours, then washed with PBS, and cultured with fresh control medium for the time indicated. [3H]-thymidine incorporation was measured at indicated times. The value of no Hcy treatment control for each time point was set as 100%, and [3H]-thymidine incorporation was divided by the control value. Values represent the mean ± SD of 3 separate experiments (n = 9). *P<.01 versus 0 hour sample in control medium after Hcy treatment.

Figure 2. Suppression of cyclin A, but not cyclin D1 and cyclin E mRNA expression by Hcy

HUVECs were exposed to 25 μM or 50 μM DL-Hcy for 30 hours (A), to 50 μM DL-Hcy or L-HH (B), and to 50 μM DL-Hcy or L-Cys (C) for the indicated times. Total cellular RNA (10 μg) was used for Northern blot analysis. The RNA blot was hybridized to human cyclin A, D1, and E probes successively, and then to an 18S oligonucleotide probe (n = 3).

Figure 3. Effect of Hcy on the protein expression and kinase activity of G₁/S cyclins and CDK2

HUVECs were exposed to 50 μg DL-Hcy or L-Cys for the indicated times. Proteins (50 μg) were analyzed by Western blotting with antibodies against cyclins A, D1, E, or CDK2 respectively (A), and by immunoprecipitation with the same antibody for associated kinase or kinase activity (B). Histone H1 (200 μg/mL) was used as phosphorylation substrate for cyclin A, E, or CDK2. GST-RB (40 μg/mL) was used as phosphorylation substrate for cyclin D1.

Figure 4. Effects of Hcy on half-life of cyclin A mRNA and gene transcription

(A) mRNA half-life; HUVECs were exposed to DL-Hcy for 24 hours followed by administration of actinomycin D (ACD; 10 μg/mL; t = 0) to stop transcription. Total cellular RNA was extracted at indicated times and used for Northern analysis. The corrected signal density was plotted as a percentage of 0 hour value against time in log scale. The values represent the means ± SD from 3 independent experiments (n = 3). (B) Nuclear run-on experiment; HUVECs were exposed to DL-Hcy for 30 hours. Equal amounts of 32P-labeled, in vitro–transcribed RNA probes from each group were hybridized to 1 μg denatured cyclin A and β-actin cDNA that had been immobilized on the nitrocellulose filters. The values represent the means ± SD from 3 independent experiments (n = 3). *P <.01 versus control.

Figure 5. Effect of Hcy on cyclin A promoter activity in vascular cells

HUVECs and RASMCs were transfected with 2 μg PGL2 plasmids containing SV40 early promoter or cyclin A promoter (−266/+205) by lipofectin transfection. Starting 24 hours after transfection, cells were treated with or without 50 μM DL-Hcy, and harvested 24 hours later. The corrected luciferase activity for each time point was divided by that of control (cyclin A −266/+205 plasmid in cells not treated with Hcy) and is presented as relative luciferase activity. Values represent the mean ± SD from 2 separate experiments (n = 6). *P <.01 versus control.

Figure 6. The effect of adenovirus-transduced cyclin A gene on DNA synthesis in Hcy-treated HUVECs

HUVECs were infected with control adenovirus (Ad vector), adenovirus expressing cyclin A (Ad cyclin A) or adenovirus expressing β-gal (Ad β-gal) at the indicated MOI for 24 hours before being challenged with Hcy. Cells were then treated with or without 50 μM of DL-Hcy in control medium for another 24 hours. (A) Western analysis; total protein (50 μg) from each sample was analyzed with antibodies against cyclin A. (B) Staining for β-gal; cells were infected with Ad β-gal at the indicated MOI and stained for β-gal. Photograph was taken with × 100 magnification. (C) Thymidine uptake; cells treated for 24 hours were metabolically labeled with 1 μCi (0.037 MBq)/mL [3H]-thymidine during the last 3 hours. [3H]-thymidine incorporation was measured in a liquid scintillation counter. □, control; ■, Hcy, 24 h. Values represent mean ± SD from 3 separate experiments from 3 wells (n = 9).

Figure 7. Flow cytometric analysis of ECs after adenovirus cyclin A infection in the presence of Hcy

(A) HUVECs were infected with adenovirus vector (Ad vector) or adenovirus expressing cyclin A (Ad cyclin A) at 100 MOI, and exposed to 50 μM DL-Hcy for 24 hours. The cells were harvested and subjected to FACS analysis of DNA content. (B) Cell-cycle distribution of HUVECs. Cell-cycle distribution was analyzed using a Multicycle software. The values represent the means ± SD from 5 independent experiments (n = 10). *P <.05 versus control.