3-Methylcholanthrene, an AhR agonist, caused cell-cycle arrest by histone deacetylation through a RhoA-dependent recruitment of HDAC1 and pRb2 to E2F1 complex

Abstract

We previously showed that treating vascular endothelial cells with 3-methylcholanthrene (3MC) caused cell-cycle arrest in the Go/G1 phase; this resulted from the induction of p21 and p27 and a decreased level and activity of the cyclin-dependent kinase, Cdk2. We further investigated the molecular mechanisms that modulate cell-cycle regulatory proteins through the aryl-hydrocarbon receptor (AhR)/Ras homolog gene family, member A (RhoA) dependent epigenetic modification of histone. AhR/RhoA activation mediated by 3MC was essential for the upregulation of retinoblastoma 2 (pRb2) and histone deacetylase 1 (HDAC1), whereas their nuclear translocation was primarily modulated by RhoA activation. The combination of increased phosphatase and tensin homolog (PTEN) activity and decreased phosphatidylinositide 3-kinase (PI3K) activation by 3MC led to the inactivation of the Ras-cRaf pathway, which contributed to pRb2 hypophosphorylation. Increased HDAC1/pRb2 recruitment to the E2F1 complex decreased E2F1-transactivational activity and H3/H4 deacetylation, resulting in the downregulation of cell-cycle regulatory proteins (Cdk2/4 and Cyclin D3/E). Co-immunoprecipitation and electrophoretic mobility shift assay (EMSA) results showed that simvastatin prevented the 3MC-increased binding activities of E2F1 proteins in their promoter regions. Additionally, RhoA inhibitors (statins) reversed the effect of 3MC in inhibiting DNA synthesis by decreasing the nuclear translocation of pRb2/HDAC1, leading to a recovery of the levels of cell-cycle regulatory proteins. In summary, 3MC decreased cell proliferation by the epigenetic modification of histone through an AhR/RhoA-dependent mechanism that can be rescued by statins.

Figure 1. 3MC-mediated inactivation of Ras and c-Raf in relation to suppression of Cdk2/4 and Cyclin D3/E1 expression in MCVECs.

(A) Cells were transiently challenged by 3MC (0 to 20 min) and then active forms of AhR, RhoA, Ras, c-Raf, and the MAPK family were analyzed using Western blot analysis of the indicated subcellular fractions. Anti-GAPDH, anti-Lamin A/C, and anti-VE-cadherin antibodies were used to verify the equivalent loading amounts of the cytosolic, nuclear, and membrane fractions, respectively. (B) Cells were treated with 3MC for a prolonged period (4 or 6 h) to investigate the effect of 3MC on cell-cycle regulatory proteins, including Cdk2/4 and Cyclin D3/E with CYP1A1 as a positive control for the AhR action of 3MC in Western blot analyses. Total RNA was extracted and analyzed, and GAPDH was used as an internal control. The data are presented as mean ± SEM for 3 independent experiments (^* P<0.05 vs. control group).

Figure 2. Effects of HDAC1/pRb2 nuclear translocation induced by 3MC in the downregulation of cell-cycle regulatory proteins by histone deacetylation.

(A) Cells were harvested and fractionated from MCVECs treated with 100 nM of 3MC at the indicated times, and were analyzed using Western blot. Anti-GAPDH, anti-Lamin A/C, and anti-VE-cadherin antibodies were used to verify the equivalent loading amounts of the cytosolic, nuclear, and membrane fractions, respectively. The open and closed arrowheads represented the hyper and hypophosphorylated pRb2, respectively. (B) The effect of 3MC on RB2 and HDAC1 subcellular localization was investigated using immunofluorescent chemical staining. Cells cultured in coverslips were challenged by 3MC for 1 h; this was followed by fixation and hybridization using anti-pRb2 and anti-HDAC1 antibodies and then a second antibody conjugated with Texas Red. In Figure 2B, red represents pRb2 or HDAC1-positive staining in the cytosol or nuclei. The identical fields were also stained using DAPI to target the nuclear position. We used 630× magnification (scale bar in white  = 50 μm) and recorded micrographs of the representative fields. (C) Cells were transfected with siHDAC1 (20 nM) overnight, prior to treatment with 3MC for 1 or 6 h. The protein levels of acetylated H3/H4, Cdk2/4, and Cyclin D3/E were assayed using Western blot. Membranes were probed using an anti-GAPDH antibody to verify equivalent loading. Three samples were analyzed in each group, and the values reported represent mean ± SEM (*P<0.05 vs. control group; ^# P<0.05 vs. 3MC treatment alone). (D) Cell numbers were counted for cells with indicated siRNA transfection followed by a 24 h of the 3MC challenge by using a hemo-cytometer. Data are presented as mean ± SEM of 3 independent experiments (*P<0.05 vs. control or siRNA group; ^# P<0.05 vs. 3MC treatment alone).

Figure 3. Essential role of AhR/RhoA activation in pRb2/HDAC1 upregulation and nuclear translocation induced by 3MC in MCVECs.

Cells were transfected with siAhR (A), DNRhoA, or CARhoA (B) overnight, with or without 3MC treatment for 1 h and separated into membrane, cytosolic and nuclear fractions. Western blot analysis was used to examine Ras/c-Raf cytosolic-membrane distribution and AhR/pRb2/HDAC1 cytosolic-nuclear distribution by cellular fractionation. The effects of DNRhoA and CARhoA in cell proliferation were assessed by counting the cell numbers using a hemo-cytometer. Data are presented as mean ± SEM of 3 independent experiments. (C) Cells received a 1 h pretreatment with a ROCK inhibitor (Y27632) or a LIMK inhibitor (BMS-5), followed by 1 h of the 3MC challenge; the cells were then harvested and partitioned into nuclear and cytosolic fractions. Phosphorylation of myosin light chain and cofilin were used as positive controls for the actions of Y27632 and BMS-5, respectively. GAPDH, Lamin A/C, and VE-cadherin were used as internal controls for the cytosol, nuclear and membrane fractions, respectively, to verify equivalent loading. Representative results of 3 separate experiments are shown, and data are presented as the mean ± SEM (*P<0.05 vs. control or DMSO group; ^# P<0.05 vs. 3MC treatment alone).

Figure 4. Involvement of PTEN and PI3K in pRb2 hypophosphorylation induced by Ras/c-Raf inactivation.

(A) Cells were pretreated with bpv (an inhibitor of PTEN) for 30 min or YS-49 (an activator of PI3K) for 1 h, followed by 1 h of 100 nM 3MC treatment. Total cell lysates, and extraction of nuclear and cytosolic proteins were analyzed using Western blot analysis for related signaling molecules involved in chromatin deaceylation. (B) Cells were transfected with a plasmid containing the DNRas gene to mimic the effect of 3MC. After 1 h of 3MC treatment, cells were harvested for Western blot analysis of the mentioned molecules in total and cytosolic-membrane extracts. GAPDH, Lamin A/C, and VE-cadherin were used as internal controls for the cytosol (or total), nuclear and membrane fractions, respectively, to verify equivalent loading. The lower panel shows the intensity of bands in the Western blots using densitometry. Data are presented as mean ± SEM of 3 independent experiments (*P<0.05 vs. control group; ^# P<0.05 vs. 3MC treatment alone).

Figure 5. 3MC treatment of MCVECs increased the formation of AhR/c-Raf/pRb2/E2F1/HDAC1 complexes, with a concomitant decrease in E2F-driven luciferase activity.

(A) Cells received 1 h of 100 nM 3MC treatment; they were then harvested with subcellular fractionation and immune-precipitated by anti-pRb2 and anti-E2F1 antibodies (2 μg) and by protein A and G agarose beads (20 μL). The resulting immunoprecipitate was detected with anti-AhR, -E2F1, -c-Raf, -HDAC1, and - pRb2 antibodies in Western blot. The samples were normalized to the IgG heavy chain, the corresponding no 3MC signal, and the 0 h time point. (B) MCVECs were transiently transfected with pGL2/E2F and pRL-TK for 24 h, as described in the “Materials and Methods” section. Luciferase activity of the reported plasmid was normalized to that of the internal control plasmid. Data were derived from 3 independent experiments and are presented as mean ± SEM (*P<0.05 vs. the control group).

Figure 6. Elimination of 3MC-mediated increases in E2F/HDAC1 binding to the E2F responsive element in the promoters of Cdk2/4 and CyclinD3/E1 by simvastatin treatment.

(A) Cells were cultured and treated with 100 nM of 3MC for 1 h after 1 h of simvastatin pretreatment. Nuclear proteins were assayed for E2F binding activity by WT and mut probes in an EMSA assay, as described in Materials and Methods. The term “100xcold” denotes a 100-fold molar excess of unlabeled oligonucleotides relative to the biotin-labeled probe; this was added to the binding assay to compete with the unlabeled oligonucleotides. The mobility of the E2F-E2F responsive element complex is indicated. Representative results of 3 experiments are shown. (B) A ChIP assay was performed in cells that received simvastatin pretreatment for 1 h, or followed by the 3MC challenge for 1 h, as indicated. The DNA associated with HDAC1 was immunoprecipitated with an anti-HDAC1 antibody; thereafter, PCR amplification was used to determine the extent of the association between HDAC1 and the functional E2F-binding sites in the promoters of Cdk2/4 and Cyclin D3/E1. An anti-GAPDH antibody was used as a negative control for the ChIP assays. Representative results of 3 experiments are shown, and data are presented as the mean ± SEM (*P<0.05 vs. the control; ^# P<0.05 vs. 3MC treatment alone).

Figure 7. Effect of statins in preventing a 3MC-mediated decrease in cell-cycle regulatory proteins and in DNA incorporation induced by RhoA inactivation.

Cells were pretreated with simvastatin or pravastatin for 1-cytosol and nuclear-cytosol of 3MC-treated MCVECs was analyzed to determine the action of statins in RhoA inactivation and their effect in preventing 3MC-mediated alterations in c-Raf/pRb2/HDAC1/histone deacetylation. (A) We analyzed fractionation after 1 h; thereafter, the levels of cell-cycle regulatory proteins were assessed by Western blot after 4 or 6 h of 3MC treatment. (B) Cells were transfected overnight with a plasmid containing DNRhoA, and 3MC treatment was administered for 6 h. A western blot analysis was conducted to examine the effect of DNRhoA on the cell-cycle regulatory proteins reduced by 3MC. GAPDH (or α-tubulin), Lamin A/C, and VE-cadherin were used as internal controls for the cytosol (or total), nuclear and membrane fractions, respectively, to verify equivalent loading. (C) Cells were pretreated with statins for 1 h; this was followed by the 3MC challenge, which was pulsed for 15 h with BrdU (Invitrogen; 0.75 μg/mL) incubation for the DNA incorporation assay. Fixed cells on coverslips were stained with a mouse anti-BrdU antibody conjugated with Texas Red. Red represents BrdU-positive staining. Identical fields were stained with DAPI (Invitrogen) to reveal the positions of cell nuclei. We recorded micrographs of the representative fields at 200× magnification (scale bar in white  = 250 μm). (D) Cell numbers were counted using a hemo-cytometer at the indicated time points in cells with various treatments. Data are presented as mean ± SEM of 3 independent experiments (*P<0.05 vs. control group; ^# P<0.05 vs. 3MC treatment alone).

Figure 8. Summary of the signal pathways of 3MC on epigenetic modification of chromatin, resulting in MCVEC cell-cycle arrest.

In the proposed signal pathways, 3MC-mediated AhR/RhoA activation modulates the PTEN/PI3K/Ras/c-Raf signaling pathways; this is followed by pRb2 hypophosphorylation, with a concomitant increase in HDAC1 upregulation and nuclear translocation. Treatment with 3MC also increased protein interaction within the E2F1 complex, including AhR, pRb2, and HDAC1. The downstream transcription factor E2F1 was negatively regulated by the co-repressor HDAC1, resulting in the downregulation of the cell-cycle regulatory proteins, including Cdk2/4 and Cyclin D3/E. Subsequently, DNA incorporation was decreased in MCVECs. The orange arrow lines indicate upregulation or activation of downstream molecules, whereas the blue lines represent downregulation or inactivation of the downstream targets. The signal pathways identified in this study are shown as solid lines with arrows, and proposed correlations are indicated by dashed lines with arrows. Additionally, the dashed-black lines indicate that the translocation occurs between cytosol and nuclei or between cytosol and the plasma membrane.