1130 NATURE MEDICINE • VOLUME 5 • NUMBER 10 • OCTOBER 1999

NEWS & VIEWS

THE CLASSIC ESTROGEN receptor signalingpathway occurs through the entry of

estrogen into the cell, translocation to thenucleus, interaction with the nuclear es-trogen receptor (ER) α or β, DNA binding,and transcriptional activation of estrogen-responsive genes. This cell signaling mech-anism can take hours or more to achieveits final downstream effects. Thus, scien-tists were puzzled by reports of rapid estro-gen effects occurring in the vasculature,breast, bone and neuronal tissue. Furtherinvestigations into estrogen signalingmechanisms led to the discov-ery of cell surface forms of es-trogen receptors that werecoupled to cytosolic signaltransduction proteins. Thesenon-genomic signaling path-ways are now known to be in-volved in regulating a widevariety of biological processes.

ERs are unique members ofthe steroid hormone receptorfamily that are regulated byphosphorylation of both tyro-sine and serine residues. Cellshave at least two forms of es-trogen receptor that originatefrom a single transcript andare localized to the cell mem-brane and to the nucleus.Recent data suggests a directlink between the surface es-trogen receptor and the mito-gen-activated protein (MAP)kinase signaling cascade.

MAP kinases are a family ofserine–threonine kinases thatbecome phosphorylated andactivated in response to a vari-ety of cell growth signals.These enzymes transduce ex-

tracellular signals from multiple mem-brane receptors to intracellular targets, in-cluding transcription factors, cytoskeletalproteins and enzymes. The MAP kinasefamily includes the extracellular-signal re-lated kinases (ERKs), which signal througha pathway involving sequential activationof Ras, Raf and mitogen-activated proteinkinase kinase (MEK). Upon binding to es-trogen, both the membrane estrogen re-

ceptors, α and β, activate ERK–relatedkinases, leading to cell proliferation1.Estrogen is known to activate the ERK sig-naling pathway in a variety of differentcell types.

In pulmonary endothelial cells estrogenhas been reported to rapidly stimulate ni-tric oxide production, which can explainits ability to induce acute dilation of bloodvessels. Chen et al.2 recently reported that17-beta-estradiol (E2) induces rapid acti-vation of endothelial nitric oxide syn-thase (eNOS) in isolated pulmonary

endothelial cells. Experimentsin isolated plasma membranesfrom pulmonary artery en-dothelial cells have demon-strated that stimulation ofeNOS by estrogen was depen-dent upon ERα. Estrogenactivation of eNOS was shown to occur through therapid activation of the MAP ki-nase pathway (Fig. 1).Complementary studies havealso shown that estrogeninduces calcium-dependenttranslocation of eNOS fromthe plasma membrane to in-tracellular sites close to the nu-cleus, an action that is againrapid (within 5 minutes), re-ceptor-mediated but non-ge-nomic3. The mechanism ofeNOS stimulation may also in-volve proteins that share bind-ing between it and theestrogen receptor, such heatshock protein (hsp) 90 (ref. 4).Thus, it has become clear thatthe short-term effects of estro-gen central to cardiovascularphysiology are mediated by

PETER COLLINS & CAROLYN WEBB

Estrogen hits the surfaceNewly discovered estrogen receptor-dependent signaling pathways demonstrate

that estrogen functions in the cytosol as well as in the nucleus.

Fig. 1 Mechanisms of rapid non-genomic receptor-dependent actionsof estrogen in four cell types. Estrogen (E) interacts with a plasma mem-brane estrogen receptor (ER). In endothelial cells this leads to the sequen-tial activation of Ras, Raf and mitogen-activated protein kinase kinase(MEK) which activates mitogen-activated protein kinase (MAPK).2

Endothelial nitric oxide synthase (eNOS) may be activated by a hetero-complex between MAPK and proteins such as heat shock protein 90(Hsp90) (ref. 4) stimulating the release of nitric oxide (NO), which resultsin relaxation of vascular smooth muscle cells. In neurons activation of c-Src and Ras by E-ER results in phosphorylation of MEK and an increase incellular MAPK, resulting in a neuroprotective effect on these cells.7 InMCF-7 breast cancer cells, E-ER interaction results in intracellular Ca++-de-pendent activation10 of the c-Src-Ras-Raf-MAPK pathway6 which may beimportant in cell cycle control. In this instance, E is acting in a similar wayto a growth factor. In oestoblasts, E-ER results in a rapid increase in MAPK,which may be involved in the control of apoptosis, cell proliferation anddifferentiation and result in bone conservation.11

E

ES

eNos

MAPK/Hsp 90

MEK

Endothelial cell

Plasmamembrane

Neuron Breast cancer cell OsteoblastVasorelaxation Neuroprotection Cell cycle stimulation Cell proliferation/

differentationBone conservation

RasRaf

MAPK

MEK

C-SrcRas

MAPK

MEK

C-SrcRas

C++Raf

MAPK

RasRaf

Bob

Crim

i

cells, telomeres may not have been com-pletely immersed in the river Styx.

1. Shay, J.W. & Bacchetti, S. A survey of telomerase ac-tivity in human cancer. Eur. J. of Cancer 33, 787–791(1997).

2. Greenberg, R.A. et al. Short dysfunctional telomeresimpair tumorigenesis in the INK4a∆2/3 cancer-pronemouse. Cell 97, 515–525.

3. Hahn, W.C. et al. Creation of human tumor cellswith defined genetic elements. Nature 400,464–472 (1999).

4. Hahn, W.C. et al. Inhibition of telomerase inhibitsthe growth of human cancer cells. Nature Med. 5,1164–1170 (1999).

5. Lingner, J. et al. Reverse transcriptase motifs in thecatalytic subunit of telomerase. Science 276,561–567 (1997).

6. Chin, L. et al. p53 deficiency rescues the adverse ef-fects of telomere loss and cooperates with telomeredysfunction to accelerate carcinogenesis. Cell 14,527–538 (1999).

7. Karlseder, J., Broccoli, D., Dai, Y., Hardy, S. & deLange, T. p53- and ATM-dependent apoptosis in-duced by telomeres lacking TRF2. Science 283,1321–1325 (1999).

8. Lee, H.-W. et al. Essential role of mouse telomerasein highly proliferative organs. Nature 392, 569–574.

9. Rudolph, K.L. et al. Longevity, stress response, andcancer in aging telomerase deficient mice. Cell 96,701–712.

10. Nugent, C., Hughes, T.R., Lue, N.F. & Lundblad, V.Cdc13 is a single-stranded telomere binding proteinwith a dual role in yeast telomere maintenance.Science 274, 249–252 (1996).

1Introgen Therapeutics, Inc

Houston, Texas 77030

Email: fL_Zumstein@introgen.com2Department of Molecular and Human Genetics

Baylor College of Medicine

Houston, Texas 77030, USA

E-mail: lundblad@bcm.tmc.edu

© 1999 Nature America Inc. • http://medicine.nature.com©

199

9 N

atu

re A

mer

ica

Inc.

• h

ttp

://m

edic

ine.

nat

ure

.co

m

NATURE MEDICINE • VOLUME 5 • NUMBER 10 • OCTOBER 1999 1131

NEWS & VIEWS

ERα functioning in a non-genomic man-ner to activate eNOS through MAP kinase-dependent mechanisms.

Acute estrogen exposure stimulates ni-tric oxide production and endothelium-de-pendent coronary dilation in femaleprimates5 and prevents coronary arterialconstriction in post-menopausal women6,whereas 20-minute exposure of male ather-oscerotic coronary arteries to estrogen doesnot enhance coronary endothelial func-tion.7 These data suggest gender specificityin some estrogen receptor signaling path-ways in enhancing endothelium-depen-dent relaxation, which may be linked tothe long-term protective effect on the de-velopment of coronary artery disease.

In neuronal cells, similar rapid activa-tion of the MAP kinase signaling pathwayby estrogen results in neuroprotection inprimary cortical neurons after glutamateexcitotoxicity (ref. 7)(Fig. 1). These neuro-protective effects of estrogen, which werereported to occur within 5 minutes afterexposure, occurred through the transientactivation of c-src-tyrosine kinases andtyrosine phosphorylation of p21(ras)-gua-nine nucleotide activating protein in anER-dependent manner. These data alsohelp explain the clinical observation thatestrogen replacement therapy aftermenopause may reduce the incidence ofAlzheimer’s disease8.

In human breast cancer cells (MCF-7cells), the MAP kinase pathway is alsorapidly activated (within 2 minutes) by es-trogen receptor complex (ref. 9)(Fig. 1).

Estradiol treatment of MCF-7 cells re-sulted in the transient activation of MAPkinases ERK 1 and ERK 2, involving Rasactivation and tyrosine phosphorylationof Shc and p190—both substrates of acti-vated c-src that once phosphorylated, in-teract with other proteins and upregulateRas. ER-dependent activation of c-src re-quired intracellular calcium mobilization,a pre-requisite for the activation of ERKsin these cancer cells10. Rapid activation ofthis pathway may be involved in the cellcycle control of these cells by estrogen.

E2 induces MAPK phosphorylation andactivation within 5 minutes in osteoblasts(ref. 11)(Fig. 1). This action is almost cer-tainly mediated through a plasma mem-brane receptor that is present on bone cells.MAPK activation by estrogen may there-fore regulate cell proliferation and differen-tiation in these cells leading to increasedbone formation.

We are now expanding the function ofsteroid hormone receptors beyond theconfines of sexual differentiation and re-productive neuroendocrine function. It isclear that the estrogen receptor is not onlya ligand-induced transcriptional en-hancer, but also a mediator of common in-tracellular signaling pathways in multiplecell types. Gaining a better understandingof the rapid actions of estrogen may leadto new therapeutic strategies for treatmentof cell proliferative, neurodegenerativeand cardiovascular defects.

1. Razandi, M., Pedram, A., Greene, G.L. & Levin, E.R.Cell membrane and nuclear estrogen receptors (ERs)

originate from a single transcript: studies of ER alphaand ER beta expressed in Chinese hamster ovarycells. Mol. Endocrinol. 13, 307–319 (1999).

2. Chen, Z. et al. Estrogen receptor alpha mediates thenongenomic activation of endothelial nitric oxidesynthase by estrogen. J. Clin. Invest. 103, 401–406(1999).

3. Goetz, R.M. et al. Estradiol induces the calcium-de-pendent translocation of endothelial nitric oxide syn-thase. Proc. Natl. Acad. Sci. USA 96, 2788–2793(1999).

4. Garcia-Cardena, G., Fan R., Shah V., et al. Dynamicactivation of endothelial nitric oxide synthase Hsp90.Nature 392, 821–824 (1998).

5. Williams, J.K., Adams, M.R., Herrington, D.M. &Clarkson, T.B. Short-term administration of estrogenand vascular responses of atherosclerotic coronaryarteries. J. Am. Coll. Cardiol. 20, 452–457 (1992).

6. Collins, P. et al. 17 beta-Estradiol attenuates acetyl-choline-induced coronary arterial constriction inwomen but not men with coronary heart disease.Circulation 92, 24–30 (1995).

7. Singer, C.A., Figueroa-Masot, X.A., Batchelor, R.H. &Dorsa, D.M. The mitogen-activated protein kinasepathway mediates estrogen neuroprotection afterglutamate toxicity in primary cortical neurons. J.Neurosci. 19 2455–2463 (1999).

8. Kawas, C. et al. A prospective study of estrogen re-placement therapy and the risk of developingAlzheimer’s disease: the Baltimore longitudinal studyof aging. Neurology 48, 1517–1521 (1997).

9. Migliaccio, A., Di DM, Castoria G., et al. Tyrosine ki-nase/p21ras/MAP kinase pathway activation byestradiol-receptor complex in MCF-7 cells. EMBO J.15, 1292–1300 (1996).

10. Improta-Brears, T. et al. Estrogen-induced activationof mitogen-activated protein kinase requires mobi-lization of intracellular calcium. Proc. Natl. Acad. Sci.USA 96, 4686–4691 (1999).

11. Endoh, H. et al. Rapid activation of MAP kinase by es-trogen in the bone cell line. Biochem. Biophys. Res.Commun. 235, 99–102 (1997).

Imperial CollegeSchool of MedicineNational Heart & Lung InstituteDovehouse Street London, SW3 6LY, UKE-mail: peter.collins@ic.nc.uk

EVERYONE HAS EXPERIENCED some form ofanxiety. The scientist entering a PhD

oral exam becomes restless and jumpy –sweating, racing heart and increasedblood pressure combine with a reluctanceto enter the ‘threatening’ situation.However, for some people, the samesymptoms can occur with a visit to thedentist or even with shopping. This is ex-cessive anxiety, which interferes withdaily life. It is one of the most commonreasons for psychiatric treatment and oneof the most costly disorders to society.

Anti-anxiety drugs (anxiolytics) areoften prescribed for anxiety disorders.‘Classical’ anxiolytics such as barbiturates,meprobamate and benzodiazepines, actthrough the A-type receptor for GABA,

the main inhibitory transmitter in thebrain. The benzodiazepines bind to the γ2subunit of GABAA-receptors, increasingtheir affinity for GABA. This increase inthe effects of GABA is anxiolytic, but it isalso sedative, hypnotic, and addictive.

Very little is known about the molecu-lar basis of anxiety disorder. However, inthe September issue of Nature Neuroscience,Crestani et al.1 describe the creation ofmice heterozygous for the GABAA-receptorγ2 subunit. These mice have reduced ben-zodiazepine binding, indicating a reduc-tion in the number of γ2 subunits, andbehave like patients suffering from anxi-

ety disorders. They avoid threats (unfamil-iar, elevated or brightly lit areas), and aremore likely to treat ambiguous cues asthreats. Like highly anxious people, the γ2heterozygous mice are more sensitive tothe anxiolytic effects of the benzodi-azepine diazepam.

Are these mice valid models of anxietyin humans? Does this gene also predis-pose people to anxiety disorders? To an-swer these questions, we must firstdistinguish between the different types ofanxiety disorders2,3,4, identify the brainsystems that control them3,4,5,6, and distin-guish vulnerability to anxiety disorderfrom the disorder itself.

Vulnerability to anxiety disorders isoften believed to reflect a ‘general neurotic

NEIL MCNAUGHTON

A gene promotes anxiety in mice—and also in scientistsA partial impairment of GABAA receptor function in mice causes behavioral changes that resemble generalized

anxiety disorder in humans. But understanding the genetic control of the ‘neurotic’ personality is still a challenge.

© 1999 Nature America Inc. • http://medicine.nature.com©

199

9 N

atu

re A

mer

ica

Inc.

• h

ttp

://m

edic

ine.

nat

ure

.co

m