I. Yuhanna - Academia.edu (original) (raw)

Papers by I. Yuhanna

Circulation, Nov 3, 2009

Although estrogen receptors (ER) function classically as transcription factors, a membrane-associ... more Although estrogen receptors (ER) function classically as transcription factors, a membrane-associated ER subpopulation initiates extranuclear responses in various cell types including endothelial cells (EC) and breast cancer cells. We investigated the basis and relevance of extranuclear ER function in endothelium using an estrogen-dendrimer conjugate (EDC) with 21 estrogen molecules stably conjugated to a large inert dendrimer that is excluded from the cell nucleus. Estradiol (E2) and EDC stimulated comparable EC growth and migration that was dependent on ER α , Src, G α i and endothelial NO synthase (eNOS) stimulation. The utility of EDC for in vivo studies was then examined. Ovariectomized female mice were given vehicle, E2, dendrimer, or EDC to yield estrogen-equivalent plasma concentrations for E2 and EDC of 10 −8M. In whole body imaging of estrogen-response-element-luciferase (ERE-Luc) reporter mice, E2 caused luciferase activation and EDC did not. Similarly, ER-regulated genes in mouse uterus were modified by E2 but not EDC. In addition, serum from EDC-treated mice did not activate ERE-Luc in HEK293 cells expressing ER α , but it stimulated nongenomic eNOS activation in EC. As such, EDC remains intact to specifically target extranuclear processes in vivo . Following perivascular electric injury of the carotid artery, E2 and EDC caused comparable 70 – 80% reendothelialization that was negated by the ER antagonist ICI 182,780 and was also absent in ER α −/− mice. In contrast, E2 elicited a uterotrophic response that was prevented by ICI 182,780, but EDC did not. Reendothelialization with E2 or EDC was prevented by pertussis toxin but not by B-oligomer. To study extranuclear ER signaling and breast cancer growth in vivo , MCF-7 cell tumor xenografts were established with E2 for 28d in SCID mice that were then treated with vehicle, E2, dendrimer or EDC for 21d. Whereas E2 caused further tumor growth, EDC did not, mimicking prior findings in cultured MCF-7 cells. Thus, EDC is a novel extranuclear selective ER modulator (SERM) in vivo , extranuclear ER α signaling is operative in vivo and promotes endothelial monolayer integrity, and these processes can potentially be harnessed to provide cardiovascular protection without increasing uterine or breast cancer risk.

Circulation …, 2000

Estrogen causes nitric oxide (NO)-dependent vasodilation due to estrogen receptor (ER) alpha-medi... more Estrogen causes nitric oxide (NO)-dependent vasodilation due to estrogen receptor (ER) alpha-mediated, nongenomic activation of endothelial NO synthase (eNOS). The subcellular site of interaction between ERalpha and eNOS was determined in studies of isolated endothelial cell plasma membranes. Estradiol (E(2), 10(-8) mol/L) caused an increase in eNOS activity in plasma membranes in the absence of added calcium, calmodulin, or eNOS cofactors, which was blocked by ICI 182,780 and ERalpha antibody. Immunoidentification studies detected the same 67-kDa protein in endothelial cell nucleus, cytosol, and plasma membrane. Plasma membranes from COS-7 cells expressing eNOS and ERalpha displayed ER-mediated eNOS stimulation, whereas membranes from cells expressing eNOS alone or ERalpha plus a myristoylation-deficient mutant eNOS were insensitive. Fractionation of endothelial cell plasma membranes revealed ERalpha protein in caveolae, and E(2) caused stimulation of eNOS in isolated caveolae that was ER-dependent; noncaveolae membranes were insensitive. Acetylcholine and bradykinin also activated eNOS in isolated caveolae. Furthermore, the effect of E(2) on eNOS in caveolae was prevented by calcium chelation. Thus, a subpopulation of ERalpha is localized to endothelial cell caveolae where they are coupled to eNOS in a functional signaling module that may regulate the local calcium environment. The full text of this article is available at http://www.circresaha.org.

Molecular endocrinology (Baltimore, Md.), 2005

Molecular Endocrinology, 2007

Circulation, Oct 31, 2006

Abstract—Estrogen causes nitric oxide (NO)-dependent vasodilation due to estrogen receptor (ER) a... more Abstract—Estrogen causes nitric oxide (NO)-dependent vasodilation due to estrogen receptor (ER) a-mediated, nongenomic,activation of endothelial NO synthase (eNOS). The subcellular site of interaction between,ERa and eNOS was determined in studies of isolated endothelial cell plasma membranes. Estradiol (E 2 ,1 0 , mol/L) caused an increase in eNOS activity in plasma membranes in the absence of added calcium, calmodulin, or

Molecular Endocrinology, 2005

Estradiol stimulates endothelial nitric oxide synthase (eNOS) via the activation of plasma membra... more Estradiol stimulates endothelial nitric oxide synthase (eNOS) via the activation of plasma membrane (PM)-associated estrogen receptor (ER) alpha. The process requires Src and erk signaling and eNOS phosphorylation by phosphoinositide 3-kinase (PI3 kinase)-Akt kinase, with Src and PI3 kinase associating with ERalpha upon ligand activation. To delineate the basis of nongenomic eNOS stimulation, the potential roles of ERalpha domains necessary for classical nuclear function were investigated in COS-7 cells. In cross-linking studies, estradiol-17beta (E2) caused PM-associated ERalpha to form dimers. However, eNOS activation by E2 was unaltered for a dimerization-deficient mutant ERalpha (ERalphaL511R). In contrast, ERalpha mutants lacking the nuclear localization signals (NLS), NLS2,3 (ERalphaDelta250-274) or the DNA binding domain (ERalphaDelta185-251), which targeted normally to PM and caveolae/rafts, were incapable of activating eNOS. The loss of NLS2/NLS3 prevented Src and erk activation, and it altered ligand-induced PI3 kinase-ERalpha interaction and prevented eNOS phosphorylation. Loss of the DNA binding domain did not change E2 activation of Src or erk, but ligand-induced PI3 kinase-ERalpha binding and eNOS phosphorylation did not occur. Thus, dimerization is not required for ERalpha coupling to eNOS; however, NLS2/NLS3 plays a role in Src activation, and the DNA binding region is involved in the dynamic interaction between ERalpha and PI3 kinase.

Molecular endocrinology (Baltimore, Md.), 2002

ERalpha and ERbeta serve classically as transcription factors, and ERalpha also mediates nongenom... more ERalpha and ERbeta serve classically as transcription factors, and ERalpha also mediates nongenomic responses to E2 such as the activation of endothelial nitric oxide synthase (eNOS). In contrast, the nongenomic capacities of endogenous ERbeta are poorly understood. We evaluated eNOS activation by E2 in cultured endothelial cells that express endogenous ERbeta to determine whether the ERbeta isoform has nongenomic action and to reveal the subcellular locale of that function. A subpopulation of ERbeta was localized to the endothelial cell plasma membrane, overexpression of ERbeta enhanced rapid eNOS stimulation by E2, and the response to endogenous ER activation was inhibited by the ERbeta-selective antagonist RR-tetrahydrochrysene (THC). eNOS activation through ERbeta was reconstituted and shown to occur independent of ERalpha in COS-7 cells, and ERbeta protein in COS-7 was directed to the plasma membrane. THC also blunted E2 activation of eNOS in isolated endothelial cell plasma me...

Molecular Endocrinology, 2013

A subpopulation of plasma membrane-associated estrogen receptor (ER)α interact directly with G pr... more A subpopulation of plasma membrane-associated estrogen receptor (ER)α interact directly with G proteins and mediate nonnuclear receptor signaling. This mechanism underlies numerous processes, including important cardiovascular protective actions of estradiol (E(2)), such as the activation of endothelial NO synthase (eNOS) and endothelial cell growth and migration. In the present work we sought a genetic approach to differentiate nonnuclear from nuclear ERα actions. We generated single alanine substitutions within the Gαi-binding domain of ERα (amino acids 251-260) and tested signaling to eNOS or ERK1,2 and activation of luciferase (Luc) reporters signifying transactivation via direct or indirect ERα-DNA binding in HeLa cells. The point mutants ERα-R256A, ERα-K257A, ERα-D258A, and ERα-R260A were all incapable of activating eNOS in response to E(2), and ERα-R256A and ERα-D258A also showed loss of ERK1,2 activation. In contrast, ERα-R256A, ERα-K257A, ERα-D258A, and ERα-R260A all displayed normal capacity to invoke E(2)-induced transactivation of estrogen response element (ERE)-Luc or Sp1-Luc. However, whereas activator protein 1-Luc activation by ERα-R256A and ERα-D258A was intact, ERα-K257A and ERα-R260A were incapable of activator protein 1-Luc activation. In in vitro pull-down assays with the two mutants that lack all nonnuclear functions tested and retain all nuclear functions tested, ERα-R256A and ERα-D258A, there was normal direct interaction between Gαi and ERα-R256A and an absence of interaction between Gαi and ERα-D258A. When expressed in endothelial cells, these two mutants prevented E(2)-induced migration and eNOS activation mediated by endogenous receptor, indicative of dominant-negative action. Thus, the point mutants ERα-R256A and ERα-D258A in the receptor GαI-binding domain provide genetic segregation of nonnuclear from nuclear ERα function.

Molecular Endocrinology, 2002

Hypertension, 2015

There is a well-recognized association between obesity, inflammation, and hypertension. Why obesi... more There is a well-recognized association between obesity, inflammation, and hypertension. Why obesity causes hypertension is poorly understood. We previously demonstrated using a C-reactive protein (CRP) transgenic mouse that CRP induces hypertension that is related to NO deficiency. Our prior work in cultured endothelial cells identified the Fcγ receptor IIB (FcγRIIB) as the receptor for CRP whereby it antagonizes endothelial NO synthase. Recognizing known associations between CRP and obesity and hypertension in humans, in the present study we tested the hypothesis that FcγRIIB plays a role in obesity-induced hypertension in mice. Using radiotelemetry, we first demonstrated that the hypertension observed in transgenic mouse-CRP is mediated by the receptor, indicating that FcγRIIB is capable of modifying blood pressure. We then discovered in a model of diet-induced obesity yielding equal adiposity in all study groups that whereas FcγRIIB(+/+) mice developed obesity-induced hypertensio...

Nature medicine, 2001

Atherosclerosis is the primary cause of cardiovascular disease, and the risk for atherosclerosis ... more Atherosclerosis is the primary cause of cardiovascular disease, and the risk for atherosclerosis is inversely proportional to circulating levels of high-density lipoprotein (HDL) cholesterol. However, the mechanisms by which HDL is atheroprotective are complex and not well understood. Here we show that HDL stimulates endothelial nitric oxide synthase (eNOS) in cultured endothelial cells. In contrast, eNOS is not activated by purified forms of the major HDL apolipoproteins ApoA-I and ApoA-II or by low-density lipoprotein. Heterologous expression experiments in Chinese hamster ovary cells reveal that scavenger receptor-BI (SR-BI) mediates the effects of HDL on the enzyme. HDL activation of eNOS is demonstrable in isolated endothelial-cell caveolae where SR-BI and eNOS are colocalized, and the response in isolated plasma membranes is blocked by antibodies to ApoA-I and SR-BI, but not by antibody to ApoA-II. HDL also enhances endothelium- and nitric-oxide-dependent relaxation in aortae ...

Circulation research, Jan 24, 2000

The American journal of physiology, 1999

The investigation of fetal pulmonary endothelial cell gene expression and function has been limit... more The investigation of fetal pulmonary endothelial cell gene expression and function has been limited by the requirement for primary cells. In an effort to establish an immortalized cell line, ovine fetal pulmonary artery endothelial cells (PAECs; passage 5) were permanently transfected with the E6 and E7 open reading frames of human papillomavirus type 16, and phenotypes related to nitric oxide (NO) production were evaluated up to passage 28. Acetylated low-density lipoprotein uptake, endothelial NO synthase (eNOS) expression, and proliferation rates were unaltered by immortalization. Acetylcholine-stimulated eNOS activity was 218-255% above basal levels in immortalized cells, and this was comparable to the 250% increase seen in primary PAECs (passage 6). eNOS was also acutely activated by estradiol to levels 197-309% above basal, paralleling the stimulation obtained in primary cells. In addition, the expression of estrogen receptor-alpha, which has recently been shown to mediate the...