Publications by authors named "Wan-Ru Lee"

18 Publications

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Vibrio deploys type 2 secreted lipase to esterify cholesterol with host fatty acids and mediate cell egress.

Elife 2020 08 18;9. Epub 2020 Aug 18.

Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, United States.

Pathogens find diverse niches for survival including inside a host cell where replication occurs in a relatively protective environment. is a facultative intracellular pathogen that uses its type 3 secretion system 2 (T3SS2) to invade and replicate inside host cells. Analysis of the T3SS2 pathogenicity island encoding the T3SS2 appeared to lack a mechanism for egress of this bacterium from the invaded host cell. Using a combination of molecular tools, we found that VPA0226, a constitutively secreted lipase, is required for escape of from the host cells. This lipase must be delivered into the host cytoplasm where it preferentially uses fatty acids associated with innate immune response to esterify cholesterol, weakening the plasma membrane and allowing egress of the bacteria. This study reveals the resourcefulness of microbes and the interplay between virulence systems and host cell resources to evolve an ingenious scheme for survival and escape.
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http://dx.doi.org/10.7554/eLife.58057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7434443PMC
August 2020

27-Hydroxycholesterol Promotes Adiposity and Mimics Adipogenic Diet-Induced Inflammatory Signaling.

Endocrinology 2019 10;160(10):2485-2494

Center for Nuclear Receptors and Cell Signaling, Department of Biology and Biochemistry, University of Houston, Houston, Texas.

27-Hydroxycholesterol (27HC) is an abundant cholesterol metabolite and has detrimental effects on the cardiovascular system, whereas its impact on adiposity is not well known. In this study, we found that elevations in 27HC cause increased body weight gain in mice fed a high-fat/high-cholesterol diet in an estrogen receptor α-dependent manner. Regardless of diet type, body fat mass was increased by 27HC without changes in food intake or fat absorption. 27HC did not alter energy expenditure in mice fed a normal chow diet and increased visceral white adipose mass by inducing hyperplasia but not hypertrophy. Although 27HC did not augment adipocyte terminal differentiation, it increased the adipose cell population that differentiates to mature adipocytes. RNA sequencing analysis revealed that 27HC treatment of mice fed a normal chow diet induces inflammatory gene sets similar to those seen after high-fat/high-cholesterol diet feeding, whereas there was no overlap in inflammatory gene expression among any other 27HC administration/diet change combination. Histological analysis showed that 27HC treatment increased the number of total and M1-type macrophages in white adipose tissues. Thus, 27HC promotes adiposity by directly affecting white adipose tissues and by increasing adipose inflammatory responses. Lowering serum 27HC levels may lead to an approach targeting cholesterol to prevent diet-induced obesity.
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http://dx.doi.org/10.1210/en.2019-00349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6760292PMC
October 2019

Clinicopathologic Characterization of GREB1-rearranged Uterine Sarcomas With Variable Sex-Cord Differentiation.

Am J Surg Pathol 2019 07;43(7):928-942

Department and Graduate Institute of Pathology, National Taiwan University Hospital, National Taiwan University College of Medicine.

Uterine mesenchymal tumors are genetically heterogenous; those with uniform cytomorphology, best exemplified by endometrial stromal tumors, often contain various fusion genes. Novel fusions involving ESR1 and GREB1, key factors in sex hormone pathways, have been implicated in rare uterine mesenchymal tumors. Particularly, the fusions between 5'-ESR1/GREB1 and 3'-NCOA2/NCOA3 were recently identified in 4 uterine tumors resembling ovarian sex-cord tumor (UTROSCT). By RNA sequencing, pathology review, and FISH screening, we identified 4 uterine sarcomas harboring rearranged GREB1, including GREB1-NCOA2 and the novel GREB1-NR4A3, GREB1-SS18, and GREB1-NCOA1, validated by RT-PCR and/or FISH. They occurred in the myometrium of postmenopausal women and were pathologically similar despite minor differences. Tumor cells were generally uniform and epithelioid, with vesicular nuclei and distinct to prominent nucleoli. Growth patterns included solid sheets, trabeculae/cords, nests, and fascicles. Only 1 tumor showed small foci of definitive sex-cord components featuring well-formed tubules, retiform structures, Leydig-like cells, and lipid-laden cells and exhibiting convincing immunoreactivity to sex-cord markers (calretinin, α-inhibin, and Melan-A). In contrast, all the 4 classic UTROSCT we collected occurred in premenopausal patients, consisted predominantly of unequivocal sex-cord elements, prominently expressed multiple sex-cord markers, and harbored ESR1-NCOA3 fusion. Combined with previously reported cases, GREB1-rearranged tumors involved significantly older women (P=0.001), tended to be larger and more mitotically active, showed more variable and often inconspicuous sex-cord differentiation, and appeared to behave more aggressively than ESR1-rearranged UTROSCT. Therefore, these 2 groups of tumors might deserve separate consideration, despite some overlapping features and the possibility of belonging to the same disease spectrum.
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http://dx.doi.org/10.1097/PAS.0000000000001265DOI Listing
July 2019

Estrogen Receptor Beta-Mediated Modulation of Lung Cancer Cell Proliferation by 27-Hydroxycholesterol.

Front Endocrinol (Lausanne) 2018 23;9:470. Epub 2018 Aug 23.

Center for Nuclear Receptors and Cell Signaling, University of Houston, Houston, TX, United States.

27-hydroxycholesterol (27HC) is an abundant cholesterol metabolite in human circulation and promotes breast cancer cell proliferation. Although lung is one of the organs that contain high levels of 27HC, the role of 27HC in lung is unknown. In this study, we found that 27HC promotes lung cancer cell proliferation in an estrogen receptor β (ERβ)-dependent manner. The expression of 27HC-generating enzyme CYP27A1 is higher in lung cancer cells than in normal lung cells. Treatment with 27HC increased cell proliferation in ERβ-positive lung cancer cells, but not in ERα-positive or ER-negative cells. The effect on cell proliferation is specific to 27HC and another oxysterol, 25-hydroxycholesterol that has a similar oxysterol structure with 27HC. Moreover, among ligands for nuclear receptors tested, only estrogen had the proliferative effect, and the effect by 27HC and estrogen was inhibited by ERβ-specific, but not ERα-specific, inhibitors. In addition, the effect by 27HC was not affected by membrane-bound estrogen receptor GPR30. Interestingly, despite the high expression of CYP27A1, endogenously produced 27HC was not the major contributor of the 27HC-induced cell proliferation. Using kinase inhibitors, we found that the effect by 27HC was mediated by the PI3K-Akt signaling pathway. These results suggest that 27HC promotes lung cancer cell proliferation via ERβ and PI3K-Akt signaling. Thus, lowering 27HC levels may lead to a novel approach for the treatment of lung cancer.
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http://dx.doi.org/10.3389/fendo.2018.00470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6116707PMC
August 2018

Cortical actin contributes to spatial organization of ER-PM junctions.

Mol Biol Cell 2017 Nov 27;28(23):3171-3180. Epub 2017 Sep 27.

Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX 75390

Endoplasmic reticulum-plasma membrane (ER-PM) junctions mediate crucial activities ranging from Ca signaling to lipid metabolism. Spatial organization of ER-PM junctions may modulate the extent and location of these cellular activities. However, the morphology and distribution of ER-PM junctions are not well characterized. Using photoactivated localization microscopy, we reveal that the contact area of single ER-PM junctions is mainly oblong with the dimensions of ∼120 nm × ∼80 nm in HeLa cells. Using total internal reflection fluorescence microscopy and structure illumination microscopy, we show that cortical actin contributes to spatial distribution and stability of ER-PM junctions. Further functional assays suggest that intact F-actin architecture is required for phosphatidylinositol 4,5-bisphosphate homeostasis mediated by Nir2 at ER-PM junctions. Together, our study provides quantitative information on spatial organization of ER-PM junctions that is in part regulated by F-actin. We envision that functions of ER-PM junctions can be differentially regulated through dynamic actin remodeling during cellular processes.
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http://dx.doi.org/10.1091/mbc.E17-06-0377DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5687020PMC
November 2017

Estrogen receptor coregulator binding modulators (ERXs) effectively target estrogen receptor positive human breast cancers.

Elife 2017 08 8;6. Epub 2017 Aug 8.

Department of Obstetrics and Gynecology, University of Texas Health Science Center, San Antonio, United States.

The majority of human breast cancer is estrogen receptor alpha (ER) positive. While anti-estrogens/aromatase inhibitors are initially effective, resistance to these drugs commonly develops. Therapy-resistant tumors often retain ER signaling, via interaction with critical oncogenic coregulator proteins. To address these mechanisms of resistance, we have developed a novel ER coregulator binding modulator, ERX-11. ERX-11 interacts directly with ER and blocks the interaction between a subset of coregulators with both native and mutant forms of ER. ERX-11 effectively blocks ER-mediated oncogenic signaling and has potent anti-proliferative activity against therapy-sensitive and therapy-resistant human breast cancer cells. ERX-11 is orally bioavailable, with no overt signs of toxicity and potent activity in both murine xenograft and patient-derived breast tumor explant models. This first-in-class agent, with its novel mechanism of action of disrupting critical protein-protein interactions, overcomes the limitations of current therapies and may be clinically translatable for patients with therapy-sensitive and therapy-resistant breast cancers.
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http://dx.doi.org/10.7554/eLife.26857DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5548489PMC
August 2017

RASSF4 controls SOCE and ER-PM junctions through regulation of PI(4,5)P.

J Cell Biol 2017 07 9;216(7):2011-2025. Epub 2017 Jun 9.

Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX

RAS association domain family 4 (RASSF4) is involved in tumorigenesis and regulation of the Hippo pathway. In this study, we identify new functional roles of RASSF4. First, we discovered that RASSF4 regulates store-operated Ca entry (SOCE), a fundamental Ca signaling mechanism, by affecting the translocation of the endoplasmic reticulum (ER) Ca sensor stromal interaction molecule 1 (STIM1) to ER-plasma membrane (PM) junctions. It was further revealed that RASSF4 regulates the formation of ER-PM junctions and the ER-PM tethering function of extended synaptotagmins E-Syt2 and E-Syt3. Moreover, steady-state PM phosphatidylinositol 4,5-bisphosphate (PI[4,5]P) levels, important for localization of STIM1 and E-Syts at ER-PM junctions, were reduced in -knockdown cells. Furthermore, we demonstrated that RASSF4 interacts with and regulates the activity of adenosine diphosphate ribosylation factor 6 (ARF6), a small G protein and upstream regulator of type I phosphatidylinositol phosphate kinases (PIP5Ks) and PM PI(4,5)P levels. Overall, our study suggests that RASSF4 controls SOCE and ER-PM junctions through ARF6-dependent regulation of PM PI(4,5)P levels, pivotal for a variety of physiological processes.
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http://dx.doi.org/10.1083/jcb.201606047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5496610PMC
July 2017

The transcriptional repressor GATAD2B mediates progesterone receptor suppression of myometrial contractile gene expression.

J Biol Chem 2017 07 2;292(30):12560-12576. Epub 2017 Jun 2.

From the Department of Biochemistry and the Department of Obstetrics and Gynecology, North Texas March of Dimes Birth Defects Center, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9038

The mechanisms whereby progesterone (P), acting via the progesterone receptor (PR), inhibits proinflammatory/contractile gene expression during pregnancy are incompletely defined. Using immortalized human myometrial (hTERT-HM) cells stably expressing wild-type PR-A or PR-B (PR), we found that P significantly inhibited IL-1β induction of the NF-κB target genes, and P-PR transrepression occurred at the level of transcription initiation and was mediated by decreased recruitment of NF-κB p65 and RNA polymerase II to and promoters. However, in cells stably expressing a PR-A or PR-B DNA-binding domain mutant (PR), P-mediated transrepression was significantly reduced, suggesting a critical role of the PR DBD. ChIP analysis of hTERT-HM cells stably expressing PR or PR revealed that P treatment caused equivalent recruitment of PR and PR to and promoters, suggesting that PR inhibitory effects were not mediated by its direct DNA binding. Using immunoprecipitation, followed by MS, we identified a transcriptional repressor, GATA zinc finger domain-containing 2B (GATAD2B), that interacted strongly with PR but poorly with PR P treatment of PR hTERT-HM cells caused enhanced recruitment of endogenous GATAD2B to and promoters. Further, siRNA knockdown of endogenous GATAD2B significantly reduced P-PR transrepression of and Notably, GATAD2B expression was significantly decreased in pregnant mouse and human myometrium during labor. Our findings suggest that GATAD2B serves as an important mediator of P-PR suppression of proinflammatory and contractile genes during pregnancy. Decreased GATAD2B expression near term may contribute to the decline in PR function, leading to labor.
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http://dx.doi.org/10.1074/jbc.M117.791350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5535031PMC
July 2017

Metabolite Regulation of Nuclear Localization of Carbohydrate-response Element-binding Protein (ChREBP): ROLE OF AMP AS AN ALLOSTERIC INHIBITOR.

J Biol Chem 2016 May 16;291(20):10515-27. Epub 2016 Mar 16.

From the Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390, the Dallas Veterans Affairs Medical Center, Dallas, Texas 75216

The carbohydrate-response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays an essential role in converting excess carbohydrate to fat storage in the liver. In response to glucose levels, ChREBP is regulated by nuclear/cytosol trafficking via interaction with 14-3-3 proteins, CRM-1 (exportin-1 or XPO-1), or importins. Nuclear localization of ChREBP was rapidly inhibited when incubated in branched-chain α-ketoacids, saturated and unsaturated fatty acids, or 5-aminoimidazole-4-carboxamide ribonucleotide. Here, we discovered that protein-free extracts of high fat-fed livers contained, in addition to ketone bodies, a new metabolite, identified as AMP, which specifically activates the interaction between ChREBP and 14-3-3. The crystal structure showed that AMP binds directly to the N terminus of ChREBP-α2 helix. Our results suggest that AMP inhibits the nuclear localization of ChREBP through an allosteric activation of ChREBP/14-3-3 interactions and not by activation of AMPK. AMP and ketone bodies together can therefore inhibit lipogenesis by restricting localization of ChREBP to the cytoplasm during periods of ketosis.
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http://dx.doi.org/10.1074/jbc.M115.708982DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4865902PMC
May 2016

PDZK1 prevents neointima formation via suppression of breakpoint cluster region kinase in vascular smooth muscle.

PLoS One 2015 17;10(4):e0124494. Epub 2015 Apr 17.

Center for Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.

Scavenger receptor class B, type I (SR-BI) and its adaptor protein PDZK1 mediate responses to HDL cholesterol in endothelium. Whether the receptor-adaptor protein tandem serves functions in other vascular cell types is unknown. The current work determined the roles of SR-BI and PDZK1 in vascular smooth muscle (VSM). To evaluate possible VSM functions of SR-BI and PDZK1 in vivo, neointima formation was assessed 21 days post-ligation in the carotid arteries of wild-type, SR-BI-/- or PDZK1-/- mice. Whereas neointima development was negligible in wild-type and SR-BI-/-, there was marked neointima formation in PDZK1-/- mice. PDZK1 expression was demonstrated in primary mouse VSM cells, and compared to wild-type cells, PDZK1-/- VSM displayed exaggerated proliferation and migration in response to platelet derived growth factor (PDGF). Tandem affinity purification-mass spectrometry revealed that PDZK1 interacts with breakpoint cluster region kinase (Bcr), which contains a C-terminal PDZ binding sequence and is known to enhance responses to PDGF in VSM. PDZK1 interaction with Bcr in VSM was demonstrated by pull-down and by coimmunoprecipitation, and the augmented proliferative response to PDGF in PDZK1-/- VSM was abrogated by Bcr depletion. Furthermore, compared with wild-type Bcr overexpression, the introduction of a Bcr mutant incapable of PDZK1 binding into VSM cells yielded an exaggerated proliferative response to PDGF. Thus, PDZK1 has novel SR-BI-independent function in VSM that affords protection from neointima formation, and this involves PDZK1 suppression of VSM cell proliferation via an inhibitory interaction with Bcr.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0124494PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4401672PMC
April 2016

The interaction between metabolism, cancer and cardiovascular disease, connected by 27-hydroxycholesterol.

Clin Lipidol 2014;9(6):617-624

Division of Pulmonary & Vascular Biology, Departments of Pediatrics & Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390, USA.

Oxysterols are metabolites of cholesterol that are produced in liver and other peripheral tissues as a means to eliminate cholesterol to bile acid. Recent studies have revealed that the most abundant circulating oxysterol 27-hydroxycholesterol (27HC) is the first identified endogenous selective estrogen receptor modulator. 27HC levels correlate well with that of cholesterol, and also rise progressively with age. 27HC affects estrogen receptor function by the antagonism of estrogen action and also by the direct modulation of the receptor function, and similar to estrogen/estrogen receptors, 27HC has many actions in various tissues. This review article introduces the recent progress in the understanding of the role of 27HC in breast cancer and cardiovascular dysfunction.
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http://dx.doi.org/10.2217/clp.14.53DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4306277PMC
January 2014

Antiphospholipid antibodies attenuate endothelial repair and promote neointima formation in mice.

J Am Heart Assoc 2014 Oct 14;3(5):e001369. Epub 2014 Oct 14.

Center for Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX (V.U., W.R.L., S.K., P.W.S., C.M.).

Background: Antiphospholipid syndrome patients have antiphospholipid antibodies (aPLs) that promote thrombosis, and they have increased cardiovascular disease risk. Although the basis for the thrombosis has been well delineated, it is not known why antiphospholipid syndrome patients also have an increased prevalence of nonthrombotic vascular occlusion. The aims of this work were to determine if aPLs directly promote medial hypertrophy or neointima formation in mice and to identify the underlying mechanisms.

Methods And Results: Medial hypertrophy and neointima formation invoked by carotid artery endothelial denudation were evaluated in mice administered normal human IgG or aPLs. While aPLs had no effect on medial hypertrophy, they caused exaggerated neointima development. This was related to an aPL-induced impairment in reendothelialization post denudation, and scratch assays in cell culture revealed that there are direct effects of aPLs on endothelium that retard cell migration. Further experiments showed that aPL antagonism of endothelial migration and repair is mediated by antibody recognition of β2-glycoprotein I, apolipoprotein E receptor 2, and a decline in bioavailable NO. Consistent with these mechanisms, the adverse impacts of aPLs on reendothelialization and neointima formation were fully prevented by the NO donor molsidomine.

Conclusions: APLs blunt endothelial repair, and there is related aPL-induced exaggeration in neointima formation after endothelial injury in mice. The initiating process entails NO deficiency mediated by β2-glycoprotein I recognition by aPLs and apolipoprotein E receptor 2. The modulation of endothelial apolipoprotein E receptor 2 function or NO bioavailability may represent new interventions to prevent the nonthrombotic vascular occlusion and resulting cardiovascular disorders that afflict antiphospholipid syndrome patients.
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http://dx.doi.org/10.1161/JAHA.114.001369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4323803PMC
October 2014

LXRβ/estrogen receptor-α signaling in lipid rafts preserves endothelial integrity.

J Clin Invest 2013 Aug 8;123(8):3488-97. Epub 2013 Jul 8.

Division of Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9063, USA.

Liver X receptors (LXR) are stimulated by cholesterol-derived oxysterols and serve as transcription factors to regulate gene expression in response to alterations in cholesterol. In the present study, we investigated the role of LXRs in vascular endothelial cells (ECs) and discovered that LXRβ has nonnuclear function and stimulates EC migration by activating endothelial NOS (eNOS). This process is mediated by estrogen receptor-α (ERα). LXR activation promoted the direct binding of LXRβ to the ligand-binding domain of ERα and initiated an extranuclear signaling cascade that requires ERα Ser118 phosphorylation by PI3K/AKT. Further studies revealed that LXRβ and ERα are colocalized and functionally coupled in EC plasma membrane caveolae/lipid rafts. In isolated aortic rings, LXR activation of NOS caused relaxation, while in mice, LXR activation stimulated carotid artery reendothelialization via LXRβ- and ERα-dependent processes. These studies demonstrate that LXRβ has nonnuclear function in EC caveolae/lipid rafts that entails crosstalk with ERα, which promotes NO production and maintains endothelial monolayer integrity in vivo.
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http://dx.doi.org/10.1172/JCI66533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3726156PMC
August 2013

Point mutations in the ERα Gαi binding domain segregate nonnuclear from nuclear receptor function.

Mol Endocrinol 2013 Jan 14;27(1):2-11. Epub 2012 Dec 14.

Division of Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

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.
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http://dx.doi.org/10.1210/me.2011-1378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3545213PMC
January 2013

Scavenger receptor class B type I is a plasma membrane cholesterol sensor.

Circ Res 2013 Jan 28;112(1):140-51. Epub 2012 Sep 28.

Department of Pediatrics, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390, USA.

Rationale: Signal initiation by the high-density lipoprotein (HDL) receptor scavenger receptor class B, type I (SR-BI), which is important to actions of HDL on endothelium and other processes, requires cholesterol efflux and the C-terminal transmembrane domain. The C-terminal transmembrane domain uniquely interacts with plasma membrane (PM) cholesterol.

Objective: The molecular basis and functional significance of SR-BI interaction with PM cholesterol are unknown. We tested the hypotheses that the interaction is required for SR-BI signaling, and that it enables SR-BI to serve as a PM cholesterol sensor.

Methods And Results: In studies performed in COS-M6 cells, mutation of a highly conserved C-terminal transmembrane domain glutamine to alanine (SR-BI-Q445A) decreased PM cholesterol interaction with the receptor by 71% without altering HDL binding or cholesterol uptake or efflux, and it yielded a receptor incapable of HDL-induced signaling. Signaling prompted by cholesterol efflux to methyl-β-cyclodextrin also was prevented, indicating that PM cholesterol interaction with the receptor enables it to serve as a PM cholesterol sensor. Using SR-BI-Q445A, we further demonstrated that PM cholesterol sensing by SR-BI does not influence SR-BI-mediated reverse cholesterol transport to the liver in mice. However, the PM cholesterol sensing does underlie apolipoprotein B intracellular trafficking in response to postprandial micelles or methyl-β-cyclodextrin in cultured enterocytes, and it is required for HDL activation of endothelial NO synthase and migration in cultured endothelial cells and HDL-induced angiogenesis in vivo.

Conclusions: Through interaction with PM cholesterol, SR-BI serves as a PM cholesterol sensor, and the resulting intracellular signaling governs processes in both enterocytes and endothelial cells.
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http://dx.doi.org/10.1161/CIRCRESAHA.112.280081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3564583PMC
January 2013

Regulation of nuclear import/export of carbohydrate response element-binding protein (ChREBP): interaction of an alpha-helix of ChREBP with the 14-3-3 proteins and regulation by phosphorylation.

J Biol Chem 2008 Sep 7;283(36):24899-908. Epub 2008 Jul 7.

Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

Carbohydrate response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in the glucose-mediated induction of gene products involved in hepatic glycolysis and lipogenesis. Glucose affects the activity of ChREBP largely through post-translational mechanisms involving phosphorylation-dependent cellular localization. In this work we show that the N-terminal region of ChREBP (residues 1-251) regulates its subcellular localization via an interaction with 14-3-3. 14-3-3 binds an alpha-helix in this region (residues 125-135) to retain ChREBP in the cytosol, and binding of 14-3-3 is facilitated by phosphorylation of nearby Ser-140 and Ser-196. Phosphorylation of ChREBP at these sites was essential for its interaction with CRM1 for export to the cytosol, whereas nuclear import of ChREBP requires dephosphorylated ChREBP to interact with importin alpha. Notably, 14-3-3 appears to compete with importin alpha for ChREBP binding. 14-3-3beta bound to a synthetic peptide spanning residues 125-144 and bearing a phosphate at Ser-140 with a dissociation constant of 1.1 microm, as determined by isothermal calorimetry. The interaction caused a shift in the fluorescence maximum of the tryptophan residues of the peptide. The corresponding unphosphorylated peptide failed to bind 14-3-3beta. These results suggest that interactions with importin alpha and 14-3-3 regulate movement of ChREBP into and out of the nucleus, respectively, and that these interactions are regulated by the ChREBP phosphorylation status.
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http://dx.doi.org/10.1074/jbc.M804308200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3259841PMC
September 2008

17beta-estradiol (E2) induces cdc25A gene expression in breast cancer cells by genomic and non-genomic pathways.

J Cell Biochem 2006 Sep;99(1):209-20

Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas 77843-4466, USA.

Cdc25A is a potent tyrosine phosphatase that catalyzes specific dephosphorylation of cyclin/cyclin-dependent kinase (cdk) complexes to regulate G1 to S-phase cell cycle progression. Cdc25A mRNA levels are induced by 17beta-estradiol (E2) in ZR-75 breast cancer cells, and deletion analysis of the cdc25A promoter identified the -151 to -12 region as the minimal E2-responsive sequence. Subsequent mutation/deletion analysis showed that at least three different cis-elements were involved in activation of cdc25A by E2, namely, GC-rich Sp1 binding sites, CCAAT motifs that bind NF-Y, and E2F sites that bind DP/E2F1 proteins. Studies with inhibitors and dominant negative expression plasmids show that E2 activates cdc25A expression through activation of genomic ERalpha/Sp1 and E2F1 and cAMP-dependent activation of NF-YA. Thus, both genomic and non-genomic pathways of estrogen action are involved in induction of cdc25A in breast cancer cells.
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http://dx.doi.org/10.1002/jcb.20902DOI Listing
September 2006

Egr-1 is activated by 17beta-estradiol in MCF-7 cells by mitogen-activated protein kinase-dependent phosphorylation of ELK-1.

J Cell Biochem 2004 Nov;93(5):1063-74

Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas 77843-4466, USA.

Early growth response-1 (Egr-1) is an immediate-early gene induced by E2 in the rodent uterus and breast cancer cells. E2 induces Egr-1 mRNA and protein levels in MCF-7 human breast cancer cells and reporter gene activity in cells transfected with pEgr-1A, a construct containing the -600 to +12 region of the Egr-1 promoter linked to the firefly luciferase gene. Deletion analysis of the Egr-1 promoter identified a minimal E2-responsive region of the promoter that contained serum response element (SRE)3 (-376 to -350) which bound Elk-1 and serum response factor (SRF) in gel mobility shift assays. Hormone-responsiveness of Egr-1 in MCF-7 cells was specifically inhibited by PD98059, a mitogen-activated protein kinase kinase inhibitor, but not by LY294002, an inhibitor of phosphatidylinositol-3-kinase (PI3-K). These results contrasted with hormone-dependent activation of the SRE in the c-fos promoter, which was inhibited by both PD98059 and LY294002. Differences in activation of the SREs in Egr-1 and c-fos were related to promoter sequence, which defines the affinities of Elk-1 and SRF to their respective binding sites. Thus, Egr-1, like c-fos, is activated through non-genomic (extranuclear) pathways of estrogen action in breast cancer cells.
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http://dx.doi.org/10.1002/jcb.20257DOI Listing
November 2004