Estrogen receptor alpha (ERα) activation functions AF-1 and AF-2 classically mediate gene transcription in response to estradiol (E2). A fraction of ERα is targeted to plasma membrane and elicits membrane-initiated steroid signaling (MISS), but the physiological roles of MISS in vivo are poorly understood. We therefore generated a mouse with a point mutation of the palmitoylation site of ERα (C451A-ERα) to obtain membrane-specific loss of function of ERα. The abrogation of membrane localization of ERα in vivo was confirmed in primary hepatocytes, and it resulted in female infertility with abnormal ovaries lacking corpora lutea and increase in luteinizing hormone levels. In contrast, E2 action in the uterus was preserved in C451A-ERα mice and endometrial epithelial proliferation was similar to wild type. However, E2 vascular actions such as rapid dilatation, acceleration of endothelial repair, and endothelial NO synthase phosphorylation were abrogated in C451A-ERα mice. A complementary mutant mouse lacking the transactivation function AF-2 of ERα (ERα-AF2 0 ) provided selective loss of function of nuclear ERα actions. In ERα-AF2 0 , the acceleration of endothelial repair in response to estrogen-dendrimer conjugate, which is a membrane-selective ER ligand, was unaltered, demonstrating integrity of MISS actions. In genome-wide analysis of uterine gene expression, the vast majority of E2-dependent gene regulation was abrogated in ERα-AF2 0 , whereas in C451A-ERα it was nearly fully preserved, indicating that membrane-to-nuclear receptor cross-talk in vivo is modest in the uterus. Thus, this work genetically segregated membrane versus nuclear actions of a steroid hormone receptor and demonstrated their in vivo tissuespecific roles.fertility | vascular effects | nongenomic effects | genomic actions A lthough estrogens classically serve as reproductive hormones, they induce cellular responses in almost all tissues in mammalian species. The biological effects of estrogens, and particularly of 17β-estradiol (E2), are initiated by their binding to intracellular estrogen receptors (ERs), ERα and ERβ, which classically serve as nuclear transcription factors (1, 2). The ERs regulate the transcription of hundreds of genes in a cell-and tissue-specific manner through their two activation functions (AFs), AF-1 and AF-2. The roles of the activation functions of ERα have been studied in vivo using mice deleted for ERαAF-1 or ERαAF-2 (3-5). These results, in particular the two models of ERαAF-2 inactivation (4, 6), suggested that many physiological functions strongly rely on nuclear ERα and gene transcription regulation. However, in addition to the nuclear, termed "genomic actions of ER," the receptors stimulate rapid (from seconds to minutes), nonnuclear signal transduction, usually termed "nongenomic" or "extranuclear" effects. The rapid mobilization of intracellular calcium and the generation of cAMP by E2 were demonstrated several decades ago (7,8). More recently, the modulation of potassium currents, phospholipase C activatio...

Abstract-Chronic increases in blood flow increase arterial diameter and NO-dependent dilation in resistance arteries.Because endothelial dysfunction accompanies metabolic syndrome, we hypothesized that flow-mediated remodeling might be impaired in obese rat resistance arteries. Obese and lean Zucker rat mesenteric resistance arteries were exposed to chronic flow increases through arterial ligation in vivo: arteries exposed to high flow were compared with normal flow arteries. Diameter was measured in vitro in cannulated arteries using pressure arteriography. After 7 days, outward remodeling (diameter increased from 346Ϯ9 to 412Ϯ11 m at 100 mm Hg) occurred in lean high-flow arteries. Endothelium-dependent tone was reduced in high-flow arteries from obese rats by contrast with lean animals. On the other hand, diameter enlargement occurred similarly in the 2 strains. The involvement of NO in endothelium-dependent dilation (evidenced by NO blockade) and endothelial NO synthase phosphorylation was smaller in obese than in lean rats. Superoxide anion and reduced nicotinamide-adenine dinucleotide phosphate oxidase subunit expression (p67phox and gp91phox) increased in obese rats and were higher in high-flow than in control arteries. Acute Tempol (a catalase mimetic), catalase plus superoxide dismutase, and L-arginine plus tetrahydrobiopterin restored endothelium-dependent dilation in obese rat normal and high-flow arteries to the level found in lean control arteries. Thus, flow-induced remodeling in obese resistance arteries was associated with a reduced endothelium-mediated dilation because of a decreased NO bioavailability and an excessive superoxide production. This dysfunction might have negative consequences in ischemic diseases in patients with obesity or metabolic syndrome. Key Words: resistance arteries Ⅲ shear stress Ⅲ NO Ⅲ reactive oxygen species Ⅲ mechanotransduction Ⅲ obesity Ⅲ metabolic syndrome T he metabolic syndrome is a common health problem. Its incidence and prevalence are increasing in parallel with the enhanced prevalence and incidence of obesity and type 2 diabetes. 1,2 This syndrome is not only a metabolic disorder; it is also associated with endothelial dysfunction 3 and vascular remodeling leading to a reduction in arterial diameter. 4 Patients with metabolic syndrome demonstrate an increasing risk of overall mortality, as well as cardiovascular morbidity and mortality. 5 Basically, an increase in blood pressure or a decrease in blood flow induces a transient adjustment in vessel diameter mediated by changes in myogenic tone and by the release of vasoactive substances. On the other hand, increasing blood flow induces a vasodilation mediated by endothelium-derived NO. A long-term exposition to altered mechanical forces induces vascular remodeling to restore tensile and shear stresses. 6,7 Long-term increase in shear stress enhances NO production by endothelial cells, and we have shown previously that NO has a key role in vascular remodeling of large 8 and resistance arteries in response to an in...

Objective-Notch3, a member of the evolutionary conserved Notch receptor family, is primarily expressed in vascular smooth muscle cells. Genetic studies in human and mice revealed a critical role for Notch3 in the structural integrity of distal resistance arteries by regulating arterial differentiation and postnatal maturation. Methods and Results-We investigated the role of Notch3 in vascular tone in small resistance vessels (tail and cerebral arteries) and large (carotid) arteries isolated from Notch3-deficient mice using arteriography. Passive diameter and compliance were unaltered in mutant arteries. Similarly, contractions to phenylephrine, KCl, angiotensin II, and thromboxane A2 as well as dilation to acetylcholine or sodium nitroprusside were unaffected. However, Notch3 deficiency induced a dramatic reduction in pressure-induced myogenic tone associated with a higher flow (shear stress)-mediated dilation in tail and cerebral resistance arteries only. Furthermore, RhoA activity and myosin light chain phosphorylation, measured in pressurized tail arteries, were significantly reduced in Notch3KO mice. Additionally, myogenic tone inhibition by the Rho kinase inhibitor Y27632 was attenuated in mutant tail arteries. Conclusions-Notch3 plays an important role in the control of vascular mechano-transduction, by modulating the RhoA/Rho kinase pathway, with opposite effects on myogenic tone and flow-mediated dilation in the resistance circulation. (Arterioscler Thromb Vasc