2-Methoxyestradiol blocks the RhoA/ROCK1 pathway in human aortic smooth muscle cells

Abstract

2-Methoxyestradiol (2-ME), a metabolite of estradiol with little affinity for estrogen receptors, inhibits proliferation of vascular smooth muscle cells; however, the molecular mechanisms underlying this effect are incompletely understood. Our previous work shows that 2-ME inhibits initiation (blocks phosphorylation of ERK and Akt) and progression (reduces cyclin expression and increases expression of cyclin inhibitors) of the mitogenic pathway and interferes with mitosis (disrupts tubulin organization). Because the RhoA/ROCK1 pathway (RhoA → ROCK1 → myosin phosphatase targeting subunit → myosin light chain) is involved in cytokinesis, herein we tested the concept that 2-ME also blocks the RhoA/ROCK1 pathway. Because of the potential importance of 2-ME for preventing/treating vascular diseases, experiments were conducted in female human aortic vascular smooth muscle cells. Microarray transcriptional profiling suggested an effect of 2-ME on the RhoA/ROCK1 pathway. Indeed, 2-ME blocked mitogen-induced GTP-bound RhoABC expression and membrane-bound RhoA, suggesting interference with the activation of RhoA. 2-ME also reduced ROCK1 expression, suggesting reduced production of the primary downstream signaling kinase of the RhoA pathway. Moreover, 2-ME inhibited RhoA/ROCK1 pathway downstream signaling, including phosphorylated myosin phosphatase targeting subunit and myosin light chain; the ROCK1 inhibitor H-1152 mimicked these effects of 2-ME; both 2-ME and H-1152 blocked cytokinesis. 2-ME also reduced the expression of tissue factor, yet another downstream signaling component of the RhoA/ROCK1 pathway. We conclude that 2-ME inhibits the pathway RhoA → ROCK1 → myosin phosphatase targeting subunit → myosin light chain, and this likely contributes to the reduced cytokinesis in 2-ME treated HASMCs.

Keywords: 2-methoxyestradiol; ROCK1; RhoA; cytokinesis; myosin light chain; myosin phosphatase targeting subunit.

Fig. 1.

Signaling schematic depicting our hypothesis of how 2-methoxyestradiol (2-ME) regulates human aortic smooth muscle cells (HASMC) cell-cycle proliferation. Extracellular mitogens activate classical signal transduction pathways that ultimately phosphorylate (and thus activate) ERK1/2 and Akt. ERK1/2 is well known to increase expression of cyclin D (G1 phase cyclin). Phosphorylated Akt activates a signal transduction pathway that stabilizes Skp2, which is the F-box protein of SCF^Skp2 ubiquitin ligase that polyubiquitinates p27^Kip1 and thus accelerates the degradation of p27^Kip1. Removal of p27^Kip1 deinhibits cyclin D activity. Cyclin D activates cyclin-dependent kinase-4 and -6 to hyperphosphorylate retinoblastoma protein (Rb), thus releasing the transcription factor E2F and allowing increased expression of downstream cyclins, resulting in the progression of the cell cycle toward mitosis. Mitosis requires the organization of tubulin to construct the mitotic spindle and achieve proper orientation of chromosomes during metaphase and their separation during anaphase. Our previous studies show that 2-ME inhibits mitogen-induced phosphorylation of both ERK1/2 and Akt and, in addition, inhibits tubulin organization (which blocks mitosis). In the current study, we propose yet a third site of action, i.e., inhibition of the RhoA/ROCK pathway, which is required for contractile ring function leading to cytokinesis. Taken together with our past results, the present study indicates that 2-ME blocks cell proliferation by interfering with mitogenesis, mitosis, and cytokinesis.

Fig. 2.

Inhibitory effects of 2-ME on PDGF-BB (20 ng/ml) induced effects in HASMCs. A: inhibitory effects of 2-ME (1 or 3 μmol/l) on cell number, DNA synthesis ([³H]thymidine incorporation), collagen synthesis ([³H]proline incorporation), and cell migration in HASMCs treated for 3 days, 24 h, 36 h, and 8 h, respectively. Results are expressed as means ± SE; n = 3 experiments in triplicates. B: representative photomicrographs and bar graph depicting concentration-dependent inhibitory effects of 2-ME on tubulin polymerization in proliferating HASMCs. Tubulin polymerization experiments were conducted in triplicate. C: time lapse phase contrast imaging of effects of 2-ME on mitogen-induced cytokinesis in HASMCs presynchronized in early S-phase by double thymidine blockade. Pictures were taken every 6 min for 24 h or until cell division. In untreated cells, cell division was accomplished within 2 h (white arrow), whereas 2-ME-treated cells failed to divide and underwent apoptotic disintegration (white arrow) after 11 h. D: modulatory effects of 2-ME on nuclear size in proliferating HASMCs treated for 5 days. Nuclear size in DAPI-stained HASMCs was assessed using the Olympus imaging system; changes are depicted in the bar graph. At least 20 cells were counted for each condition. *P < 0.05 vs. vehicle-treated control.

Fig. 3.

Photomicrographs showing the process of cell division of HASMCs in growth medium (A), with 5 µmol/l 2-ME (B), and with 1 µmol/l H-1152 (C). Cells were grown in 6-well plates and synchronized in early S-phase by double thymidine block. After the second release, cells were treated and placed under the microscope in cell culture conditions. Images were taken automatically every 6 min for 12 h.

Fig. 4.

A: effects of ROCK1 inhibitor H-1152 (1–3 μmol/l) on PDGF-BB-induced proliferation (cell number) of HASMCs. Data are means ± SE; n = 3 experiments in triplicates. B: microarray analysis of transcriptional changes in HASMCs treated with 2-ME (3 μmol/l) for 30 h. Expression levels are depicted as color scale from red (upregulation) to blue (downregulation). Gene Set Enrichment Analysis (GSEA) revealed upregulation of key RhoA/ROCK1 pathway-associated transcripts. C: significant inhibitory effects of 2-ME (3 μmol/l) on FCS (5%)-induced expression of GTP-bound RhoABC in HASMCs. D: inhibitory effects of 2-ME on PDGF-BB (20 ng/ml)-induced membrane-bound RhoA (m-RhoA) expression in HASMCs. Data are presented as means ± SE; n = 3. *P < 0.05 vs. vehicle-treated control; ^§significant inhibition vs. mitogen-treated cells.

Fig. 5.

A: time-dependent stimulatory effects of PDGF-BB (20 ng/ml) on expression of ROCK1 and inhibitory effects of 2-ME and H-1152 (ROCK1 inhibitor) on PDGF-BB-induced expression of ROCK1 in HASMCs. Data are presented as means ± SE; n = 3. *P < 0.05 vs. vehicle treated control. B: time-dependent stimulatory effects of PDGF-BB (20 ng/ml) on phosphorylation of MYPT and inhibitory effects of 2-ME and H-1152 on PDGF-BB-induced phosphorylation of myosin-targeting subunit of myosin light-chain phosphatase (MYPT) in HASMCs. C: inhibitory effects of 2-ME on angiotensin II-induced phosphorylated MYPT expression in HASMCs. Data are presented as means ± SE; n = 3.

Fig. 6.

A: time-dependent stimulatory effects of PDGF-BB (20 ng/ml) on phosphorylation of rMLC and inhibitory effects of 2-ME and H-1152 (ROCK1 inhibitor) on PDGF-BB-induced phosphorylation of regulatory subunit of myosin light chain (rMLC) in HASMCs. Data are presented as means ± SE; n = 3. *P < 0.05 vs. vehicle-treated control; ^§significant inhibition vs. mitogen-treated cells. B: representative fluorescent staining of HASMCs for inhibitory effects of 2-ME or H-1152 on PDGF-BB (20 ng/ml)-induced phosphorylation of rMLC (green fluorescence) and internal control nonphosphorylated rMLC (pink fluorescence). Three separate experiments were conducted, each in triplicate.

Fig. 7.

Western blots and bar graph (changes in optical density; means ± SE) depicting effects of 2-ME on cyclin D1, cyclin B1, phosphorylated (p)-ERK1/2, total ERK1/2, p-Akt and total Akt. Data are presented as means ± SE; n = 3. *P < 0.05 vs. vehicle-treated control; ^§significant inhibition vs. mitogen-treated cells.

Fig. 8.

Concentration-dependent inhibitory effects of 2-ME on thrombin (4 U/ml)-induced expression of tissue factor (TF) in HASMCs.

Fig. 9.

A: representative Western blot demonstrating inhibitory effects of 2-ME on thrombin (Thr; 4 U/ml)-induced TF, ROCK1, p-rMLC, and p-MYPT expression in HASMCs. Similar effects were observed in 3 separate experiments. B: stimulatory effects of PDGF-BB (20 ng/ml), thrombin (4 U/ml), TNF-α (10 ng/ml), and FCS (5%) on TF expression in cultured HASMCs, and inhibitory effects of 2-ME (1 μmol/l) on PDGF-BB, TNF-α, and FCS-induced expression of TF in cultured HASMCs. C: Inhibitory effects of 2-ME on TF and ROCK1 protein expression in rat carotids in vivo and on neointima formation following balloon injury. Rats were treated with placebo or 2-ME (350 μg·kg⁻¹·day⁻¹ iv via osmotic pumps, n = 4) and were euthanized on day 8. Carotid arteries were snap-frozen in liquid nitrogen, subsequently lysed, and proteins analyzed using Western blotting. Data are presented as means ± SE; n = 4. *P < 0.05 vs. controls.