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.2-methoxyestradiol; RhoA; ROCK1; myosin phosphatase targeting subunit; myosin light chain; cytokinesis 2-METHOXYESTRADIOL (2-ME) is an endogenous metabolite of estradiol that attenuates vascular smooth muscle cell (VSMC) proliferation, migration, and extracellular matrix synthesis (5,8,9) and reduces injury-induced neointima formation (4), cholesterol-induced atherosclerosis (9), monocrotaline-induced vascular thickening in pulmonary hypertension (9), and injuryinduced glomerosclerosis (9). Although 2-ME inhibits VSMC proliferation and is thus effective against multiple proliferative disorders (4, 9, 10), the mechanisms via which 2-ME mediates these actions remain incompletely understood.Our previously published work shows that in human aortic vascular smooth muscle cells (HASMCs) 2-ME inhibits cell proliferation in part by blocking initiation of the mitogenic pathway (4). Specifically, 2-ME inhibits phosphorylation of both ERK1/2 and Akt (Fig. 1), which turns off the mitogenic pathway by decreasing the expression and activity of cyclins. Indeed, we (4) found that 2-ME 1) blocks cell-cycle progression in both G0/G1 and in G2/M phases, 2) reduces cyclin D1 and cyclin B1 expression, 3) inhibits Cdk-1 and Cdk-2 activity, 4) reduc...

2-Methoxyestradiol Reduces Angiotensin II–Induced Hypertension and Renal Dysfunction in Ovariectomized Female and Intact Male Mice

“…A recent study showed that 2-ME inhibits serum-induced vascular smooth muscle cell (VSMC) migration and proliferation by blocking the RhoA/ROCK1 pathway both by inhibiting the activation of RhoA and by reducing the expression of ROCK1. 43 Whether 2-ME mediates the effect of estradiol to protect against Ang II-induced hypertension, cardiac fibrosis, renal dysfunction, and end-organ damage in OVX and Cyp1b1 − / − female mice and in Cyp1b1 +/+ male mice by inhibiting the RhoA/ROCK11 pathway remains to be determined. Oxidative stress is known to contribute to Ang II-induced hypertension and its pathogenesis in various models of hypertension including in Cyp1b1 − / − and OVX Cyp1b1 +/+ mice.…”

2-Methoxyestradiol causes matrix metalloproteinase 9-mediated transactivation of epidermal growth factor receptor and angiotensin type 1 receptor downregulation in rat aortic smooth muscle cells

“…Our study has demonstrated that 2ME2 binding to GPR30 mediated transactivation of EGFR and activation of ERK1/2 in RASMCs. 2ME2 is an antiproliferative agent that has been shown to lead to cell cycle arrest in human aortic smooth muscle cells through the RhoA/ROCK1 pathway (59). In our study 2ME2 transactivates EGFR that phosphorylates the MAP-kinase/ERK1/2 pathway, which does not show increased cell proliferation, although EGFR/MAPK/ ERK is an established growth promoting signaling pathway.…”

DPP4 Inhibition, NPY1-36, PYY1-36, SDF-1α, and a Hypertensive Genetic Background Conspire to Augment Cell Proliferation and Collagen Production: Effects That Are Abolished by Low Concentrations of 2-Methoxyestradiol

“…Our previous studies show that 17bestradiol, the major ovarian estrogen, is a potent inhibitor of CF, GMC, and vascular smooth muscle cell proliferation induced by serum. Moreover, these effects of 17b-estradiol are mediated by the conversion of 17b-estradiol to 2ME (Dubey et al, 1998(Dubey et al, , 2000(Dubey et al, , 2003a(Dubey et al, ,b, 2004(Dubey et al, , 2007Xiao et al, 2001;Zacharia et al, 2001Zacharia et al, , 2002Zacharia et al, , 2003Barchiesi et al, 2002Barchiesi et al, , 2006Barchiesi et al, , 2010Dubey and Jackson, 2009;Rigassi et al, 2015), a major nonestrogenic metabolite of 17b-estradiol. Therefore, it is conceivable that because of endogenous 2ME, premenopausal women are resistant to the growthpromoting effects of DPP4 inhibition, NPY 1-36 , SDF-1a, or their combination.…”