Abstract-17␤-Estradiol (E 2 ) is a rapid activator of endothelial nitric oxide synthase (eNOS). The product of this activation event, NO, is a fundamental determinant of cardiovascular homeostasis. We previously demonstrated that E 2 -stimulated endothelial NO release can occur without an increase in cytosolic Ca 2ϩ . Here we demonstrate for the first time, to our knowledge, that E 2 rapidly induces phosphorylation and activation of eNOS through the phosphatidylinositol 3 (PI3)-kinase-Akt pathway. E 2 treatment (10 ng/mL) of the human endothelial cell line, EA.hy926, resulted in increased NO production, which was abrogated by the PI3-kinase inhibitor, LY294002, and the estrogen receptor antagonist ICI 182,780. E 2 stimulated rapid Akt phosphorylation on serine 473. As has been shown for vascular endothelial growth factor, eNOS is an E 2 -activated Akt substrate, demonstrated by rapid eNOS phosphorylation on serine 1177, a critical residue for eNOS activation and enhanced sensitivity to resting cellular Ca 2ϩ levels. Adenoviral-mediated EA.hy926 transduction confirmed functional involvement of Akt, because a kinase-deficient, dominant-negative Akt abolished E 2 -stimulated NO release. The membrane-impermeant E 2 BSA conjugate, shown to bind endothelial cell membrane sites, also induced rapid Akt and consequent eNOS phosphorylation. Thus, engagement of membrane estrogen receptors results in rapid endothelial NO release through a PI3-kinase-Akt-dependent pathway. This explains, in part, the reduced requirement for cytosolic Ca 2ϩ fluxes and describes an important pathway relevant to cardiovascular pathophysiology. Key Words: estrogen Ⅲ endothelial nitric oxide synthase Ⅲ Akt Ⅲ membrane receptor E ndogenous and exogenous estrogen in premenopausal and postmenopausal women, respectively, is protective against the development of atherosclerotic cardiovascular disease. 1,2 The relevant biological effects of estrogen are numerous and include improvements in lipid and lipoprotein profiles as well as endothelial-dependent vasodilation stimulated by estrogen administered at physiological concentrations. Reports have described significant estrogen-stimulated increases in bioavailable NO. [3][4][5] Because the antiatherogenic properties of NO are emerging, it has been proposed that the cardiovascular protective effect of estrogen is mediated through augmentation of endothelial NO production. Using a human endothelial cell (EC) in vitro model, we have previously shown that 17␤-estradiol (E 2 ) induces endothelial NO release within minutes, is estrogen receptor (ER)-dependent and gene transcription-independent, and is the result of activation of endothelial nitric oxide synthase (eNOS). 6 The regulation of eNOS activity is multifaceted. This includes regulated palmitoylation and myristoylation, which are required for eNOS partitioning into membrane caveolae and consequent function. 7-10 A variety of cofactors are required for enzymatic function, including Ca 2ϩ , calmodulin, and tetrahydrobiopterin. 11,12 Recently, heat shock p...

17␤-Estradiol activates endothelial nitric oxide synthase (eNOS),The cardioprotective effects of estrogen are diverse, including both rapid non-genomic and delayed genomic effects on the blood vessel wall (reviewed in Ref. 1). Specific, rapid vascular effects, such as moderation of vasomotor tone, have been linked to an estrogen-stimulated increase in bioavailable nitric oxide (NO) 1 (2-4). 17␤-estradiol (E2) treatment of human endothelial cells (EC) induces rapid release of NO by estrogen receptor (ER)-dependent activation of endothelial nitric oxide synthase (eNOS) (5). Many factors regulate eNOS enzyme activity, including fatty acid modification, subcellular localization, and binding to numerous proteins and cofactors, including calmodulin, caveolin-1, the 90-kDa heat shock protein (HSP90), and tetrahydrobiopterin (see Ref. 6 for review). eNOS is a Ca 2ϩ / calmodulin-dependent enzyme, the activity of which is also regulated by phosphorylation. Specific phosphorylation of eNOS by the serine/threonine kinase Akt renders the enzyme more active at much lower Ca 2ϩ concentrations (7,8). We demonstrated previously that the ER-dependent activation of eNOS occurs at resting Ca 2ϩ concentrations and requires activation of the phosphatidylinositol-3-OH kinase (PI3-kinase)/ Akt pathway (9). The regulatory subunit of PI3-kinase, P85, acts to stabilize and inhibit the catalytic activity of PI3-kinase. Recently, ER was shown to specifically bind to P85 in vitro (10). The E2-induced association correlated with increases in PI3-kinase activity in EC. However, the specific mechanism for E2 activation of PI3-kinase is not known.Evidence is emerging that membrane forms of steroid hormone receptors exist and participate in signaling pathways (11)(12)(13)(14). The activity of E2 at the cell membrane has been shown in EC, neurons, and breast cancer cell lines. We previously determined that rapid E2 activation of eNOS and MAP kinase occurs through a membrane-associated ER (9, 12). The EC line EAhy.926 used in these experiments exhibits rapid E2-induced signaling but is unable to stimulate ER-dependent gene transactivation. Additionally, EAhy.926 cells do not express the traditional 66-kDa ER␣ or ER␤ but express a 46-kDa protein immunoreactive with C-terminal ER antibodies. Recently, a protein of similar size reactive with E2 and anti-ER antibodies was found to be associated with the plasma membrane in MCF-7 cells (13,14). Additionally, a 46-kDa putative ER, reactive with anti-ER antibodies, was found in wild-type and in the initial ER␣ knockout mice. This form of the receptor was thought to be responsible for E2 enhancement of basal NO production in the initial ER␣ knockout mice, because this E2 effect was lost in the complete ER␣ knockout mouse (15). In human ECs expressing both the 66-and the 46-kDa receptor, both rapid signaling to MAP kinase and gene transactivation of estrogen-responsive element-luciferase reporter was stimulated with E2 treatment (12). As previously indicated, the specific mechanism of membrane-associated ER ...

Recently a number of nonclass I genes were discovered in the human MHC class I region. One of these, FAT10, encodes a protein consisting of two domains with homology to ubiquitin. FAT10 mRNA is expressed constitutively in some lymphoblastoid lines and dendritic cells and in certain other cells after ␥-interferon induction. FAT10 protein expression is controlled at several levels including transcription, translation, and protein stability. Yeast two-hybrid screening of a human lymphocyte library and immunoprecipitation studies revealed that FAT10 noncovalently associated with MAD2, a protein implicated in a cell-cycle checkpoint for spindle assembly during anaphase. Thus, FAT10 may modulate cell growth during B cell or dendritic cell development and activation.

Aberrant blood vessel formation contributes to a wide variety of pathologies, and factors that regulate angiogenesis are attractive therapeutic targets. Endothelial and smooth muscle cell-derived neuropilin-like protein (ESDN) is a neuropilin-related transmembrane protein expressed in ECs; however, its potential effect on VEGF responses remains undefined. Here, we generated global and EC-specific Esdn knockout mice and demonstrated that ESDN promotes VEGF-induced human and murine EC proliferation and migration. Deletion of Esdn in the mouse interfered with adult and developmental angiogenesis, and knockdown of the Esdn homolog (dcbld2) in zebrafish impaired normal vascular development. Loss of ESDN in ECs blunted VEGF responses in vivo and attenuated VEGF-induced VEGFR-2 signaling without altering VEGF receptor or neuropilin expression. Finally, we found that ESDN associates with VEGFR-2 and regulates its complex formation with negative regulators of VEGF signaling, protein tyrosine phosphatases PTP1B and TC-PTP, and VEcadherin. These findings establish ESDN as a regulator of VEGF responses in ECs that acts through a mechanism distinct from neuropilins. As such, ESDN may serve as a therapeutic target for angiogenesis regulation.

Lymphokine gene expression is a precisely regulated process in T cell-mediated immune responses. In this study we demonstrate that engagement of the β2 integrin LFA-1 in human peripheral T cells markedly extends the half-life of TNF-α, GM-CSF, and IL-3 mRNA, as well as a chimeric β-globin mRNA reporter construct containing a strongly destabilizing class II AU-rich element from the GM-CSF mRNA 3′-untranslated region. This integrin-enhanced mRNA stability leads to augmented protein production, as determined by TNF-α ELISPOT assays. Furthermore, T cell stimulation by LFA-1 promotes rapid nuclear-to-cytoplasmic translocation of the mRNA-stabilizing protein HuR, which in turn is capable of binding an AU-rich element sequence in vitro. Abrogation of HuR function by use of inhibitory peptides, or marked reduction of HuR levels by RNA interference, prevents LFA-1 engagement-mediated stabilization of T cell TNF-α or IFN-γ transcripts, respectively. Thus, HuR-mediated mRNA stabilization, stimulated by integrin engagement and controlled at the level of HuR nuclear export, is critically involved in T cell activation.