Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-10-06
2003-05-20
Kemmerer, Elizabeth (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S006120, C436S501000
Reexamination Certificate
active
06566081
ABSTRACT:
BACKGROUND OF THE INVENTION
Estrogens elicit endothelium-dependent relaxation, apparently through activation of endothelial nitric oxide synthase, but the precise signaling pathway by which estrogens stimulate endothelial nitric oxide synthase (eNOS) activity is unknown. Here it is shown that the estrogen receptor (ER)-&agr; interacts directly with phosphatidylinositol (PI) 3-kinase and that estrogen-induced eNOS activity is mediated via a novel non-nuclear mechanism involving the activation of the PI3-kinase/Akt pathway. The role of this pathway in modulation of physiologic conditions mediated by the estrogen and other receptors is disclosed and claimed.
BACKGROUND OF THE INVENTION
Recent epidemiological studies suggest that the use of hormone replacement therapy (HRT) by postmenopausal women is associated with improved outcomes from cardiovascular events.
1-4
Although some of the effects of HRT are related to the beneficial changes in lipid profile
5
, other hemostatic effects such as the reduction in serum fibrinogen and plasminogen activator-inhibitor (PAI)-1 concentrations may also play an important role.
2,6
Furthermore, there is increasing evidence suggesting that a major part of estrogen's protective actions is due to its direct effects on the vascular wall. However, despite extensive research over the past few years, little information is available regarding the molecular mechanisms by which estrogens exert their protective effects.
Estrogens elicit endothelium-dependent vasodilation in animals
7,8
and humans
9-13
through the release of endothelium-derived nitric oxide (NO).
14
Estrogen stimulates NO release by increasing the activity and expression of Type III endothelial NO synthase (eNOS).
15,16
Although the activation of eNOS activity by estrogen occurs rapidly and involves the estrogen receptor (ER)-&agr; and the mitogen-activated protein (MAP) kinase pathway, the signaling mechanism has yet to be fully established.
17
Recent studies suggest that stimuli of NO release such as bradykinin and laminar shear stress increase eNOS activity via phosphorylation of eNOS protein.
18,19
In particular, the phosphorylation of eNOS in response to laminar shear stress occurs via the PI3-kinase/Akt pathway.
20,21
Interestingly, another activator of the PI3-kinase/Akt pathway, insulin, has also been shown to increase eNOS activity.
22,23
The PI3-kinase is a critical mediator of the cellular effects of many growth factors, such as platelet-derived growth factor (PDGF)
26
, insulin
22
as well as vascular endothelial growth factor (VEGF)
27
. The PI3-kinase is a heterodimeric phosphoinositide kinase composed of an 85 kD (p85) adapter/regulatory subunit and a 110 kD (p110) catalytic subunit.
24
The PI3-kinase catalyzes the synthesis of phosphatidylinositol 3,4-biphosphate (PIP
2
) and phosphatidylinositol 3,4,5-triphosphate (PIP
3
).
25
These lipid mediators act as second messengers which activate proteins containing specific PIP
3
-binding or pleckstrin homology (PH) domains. For example, the increase in intracellular PIP
3
and PIP
2
leads to the activation of phosphatidyl-dependent protein kinases, such as phosphatidyl-dependent kinase (PDK)-1 and -2.
28,29
The PDKs, in turn, selectively phosphorylate two threonine and serine residues of another important serine-threonine kinase called protein kinase B (PKB) or Akt. The activation of Akt has recently been shown to mediate many of the downstream cellular effects of PI3-kinase.
30,31
This invention disclosure reveals that estrogens increase eNOS activity through the activation of the PI3-kinase/Akt pathway. Accordingly, this invention comprehends methods wherein the molecular signaling pathway by which estrogens activate eNOS is modulated, including interactions of ER-&agr; with PI3-kinase.
SUMMARY OF THE INVENTION
Human endothelial cells were stimulated with estrogens and subsequent changes in PI3-kinase and eNOS activity were measured. The 17&bgr;-, but not 17&agr;-, estradiol (E
2
) increased PI3-kinase and eNOS activity in a time-dependent manner with maximum effect occurring 15-20 min after stimulation. The maximal effects of E
2
were completely blocked by the estrogen receptor antagonist, ICI 182,780, and a selective PI3-kinase inhibitor, wortmannin, but were only minimally affected by the mitogen-activating protein (MAP) kinase inhibitor, PD98059, or by the tyrosine kinase inhibitor, genistein. Co-immunoprecipitation studies demonstrated that the regulatory subunit of PI3-kinase, p85&agr;, associates with the estrogen receptor (ER)-&agr;, and that stimulation with E
2
increased both the amount and activity of co-immunoprecipitated PI3-kinase. The increase in PI3-kinase activity by E
2
corresponded temporally to the threonine phosphorylation of Akt and increase in eNOS activity.
Accordingly, this invention disclosure shows that the PI3-kinase/Akt signaling pathway mediates estrogen-induced activation of eNOS. Although the estrogen receptor is generally thought to function at the nuclear level, the present invention reveals that the activation of eNOS involves the direct association of ER-&agr; with PI3-kinase p85&agr;. These findings define a novel estrogen receptor signaling pathway in vascular endothelial cells and therapeutic strategies for treating vascular and non-vascular disorders.
Thus, it is one object of this invention to provide methods and compounds useful in modulating the level of activation of endothelial nitric oxide synthase.
Another object of this invention is to provide methods and compounds for modifying the effect of estrogen and related hormones on the level of PI3-kinase activity and the level of Akt activation.
Another object of this invention is to provide methods and compounds for modifying the interaction between the estrogen receptor, and related receptors, and the PI3-kinase.
Further objects and advantages of this invention will become apparent from a review of the complete disclosure and the claims appended to this invention disclosure.
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Whitfield et al. Steroid hormone receptor: evolution, ligands and molecular basis of biological function. J. Cell. Biochem. Suppls. 32/33:110-122, 1999.*
Hall et al. The multifaceted mechanisms of estradiol and estrogen receptor signaling. J. Biol. Chem. 276:36869-36872,2001.*
Simoncini,, et al., “Activation of Estrogen Receptor-&agr; Increases Endothelial Nitric Oxide Synthase Activity via the Phosphatidylinositol 3-Kinase-Akt Pathway”, Circulation / Supplement, vol. 100, No. 18, p. I.337, Nov. 2, 1999.
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Ui, et al., “Wortmannin as a unique probe for an intracellular signalling protein, phosphoinositide 3-kinase”, TIBS, vol. 20, No. 8, pp. 303-307, Aug. 1995.
Kennedy, et al., “The PI-kinase/Akt signaling pathway delivers an anti-apototic signal”, Genes and Development, vol. 11, No. 6, pp. 701-713, 1997.
Dodge, et al., “17&bgr;-Hydroxywortmannin: A Potent Inhibitor of Bone Resorption And Phosphatidylinositol-3-Kinase”, Bioorganic and Medicinal Chemistry Letters, vol. 5, No. 15, pp. 1713-1718, Aug. 3, 1995.
Marte, et al., “PKB/Akt: connecting phosphoinositide 3-kinase to cell survival and beyond”, TIBS, vol. 22, No. 9, pp. 355-358, Sep. 1, 1997.
Haynes, et al., “Membrane Estrogen Receptor Engagement Activates Endothelial Nitric Oxide Sunthase via the PI3-Kinase-Akt Pathway in Human Endothelial Cells”, Circulation Research, vol. 8, No. 7, pp. 677-682,
Chin William
Liao James K.
Kemmerer Elizabeth
Li Ruixiang
The Brigham and Women's Hospital Inc.
Wolf Greenfield & Sacks P.C.
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