Src Kinase Mediates Phosphatidylinositol 3-Kinase/Akt-dependent Rapid Endothelial Nitric-oxide Synthase Activation by Estrogen

Abstract

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 ...

Cooperative action of tamoxifen and c-Src inhibition in preventing the growth of estrogen receptor–positive human breast cancer cells

“…Previous reports have shown that c-Src and the ERa interact in a protein complex (16,18). Therefore, we first sought to determine if the K303R-ERa mutation alters the ability of ERa to bind with c-Src.…”

“…It was later shown that estrogen stimulation can activate a number of other classic signal transduction pathways, such as phosphatidylinositol 3-kinase and extracellular signal-regulated kinase 1/2 (16,17), and that ligand-induced ERa activation recruited a c-Src/p85/ERa complex (16,18). In addition to ERa signaling to c-Src, c-Src can phosphorylate ERa on tyrosine 537 (19).…”

17‐β‐Estradiol‐mediated activation of extracellular‐signal regulated kinase, phosphatidylinositol 3‐kinase/protein kinase B‐Akt and N‐methyl‐d‐aspartate receptor phosphorylation in cortical synaptoneurosomes

“…Song et al 2005), while E2 treatment also initiates interaction between ERa, Src and p85, the regulatory subunit of PI3-K (Simoncini et al 2000;Castoria et al 2001). In addition, activation of both ERK and Akt by E2 can be prevented by Src-family kinase inhibitors (PP1 or PP2), which subsequently lead to the blockade of ERK and Akt phosphorylation (Singer et al 1999;Castoria et al 2001;Haynes et al 2003).…”

Estrogen, Heat Shock Proteins, and NFκB in Human Vascular Endothelium

“…[25][26][27][28][29] An example of this is the activation of endothelial nitric oxide synthase (eNOS), which occurs through an IP3-kinase-dependent pathway and is independent of transcription. 9,30 HSP90 is involved in this, because it complexes with eNOS in the cell, and estrogen increases the binding eNOS by HSP90. The exact mechanisms through which this occurs are still being elucidated.…”