TY - JOUR
T1 - Quantitative measurement of estrogen-induced ERK 1 and 2 activation via multiple membrane-initiated signaling pathways
AU - Bulayeva, Nataliya N.
AU - Gametchu, Bahiru
AU - Watson, Cheryl S.
N1 - Funding Information:
This work was supported by NIEHS grant no. 010987. We are grateful for the skilled editing and scientific comments of Dr. David Konkel.
PY - 2004/3
Y1 - 2004/3
N2 - Estradiol (E2) and other steroids have recently been shown to initiate various intracellular signaling cascades from the plasma membrane, including those stimulating mitogen-activated protein kinases (MAPKs), and particularly extracellular-regulated kinases (ERKs). In this study we demonstrated the ability of E2 to activate ERKs in the GH3/B6/F10 pituitary tumor cell line, originally selected for its enhanced expression of membrane estrogen receptor-α (mERα). We compared E2 to its cell-impermeable analog (E2 conjugated to peroxidase, E 2-P), and to the synthetic estrogen diethylstilbestrol (DES). Time-dependent ERK activation was quantified with a novel fixed cell-based immunoassay developed to efficiently determine activation by multiple compounds over multiple parameters. Both E2 and DES produced bimodal responses, but with distinctly different time courses of enzyme phosphorylation (activation) and inactivation; E2-P induced a monophasic ERK activation. E2 also phosphorylated ERKs in concentration-dependent manner with two concentration optima (10-14 and 10-8M). Inhibitors were employed to determine pathway (ER, EGFR, membrane organization, PI3 kinase, Src kinase, Ca2+) involvement and timing of pathway activations; all affected ERK activation as early as 3-6min, suggesting simultaneous, not sequential, activation. Therefore, E2 and other estrogenic compounds can produce rapid ERK phosphorylations via nongenomic pathways, using more than one pathway for signal generation.
AB - Estradiol (E2) and other steroids have recently been shown to initiate various intracellular signaling cascades from the plasma membrane, including those stimulating mitogen-activated protein kinases (MAPKs), and particularly extracellular-regulated kinases (ERKs). In this study we demonstrated the ability of E2 to activate ERKs in the GH3/B6/F10 pituitary tumor cell line, originally selected for its enhanced expression of membrane estrogen receptor-α (mERα). We compared E2 to its cell-impermeable analog (E2 conjugated to peroxidase, E 2-P), and to the synthetic estrogen diethylstilbestrol (DES). Time-dependent ERK activation was quantified with a novel fixed cell-based immunoassay developed to efficiently determine activation by multiple compounds over multiple parameters. Both E2 and DES produced bimodal responses, but with distinctly different time courses of enzyme phosphorylation (activation) and inactivation; E2-P induced a monophasic ERK activation. E2 also phosphorylated ERKs in concentration-dependent manner with two concentration optima (10-14 and 10-8M). Inhibitors were employed to determine pathway (ER, EGFR, membrane organization, PI3 kinase, Src kinase, Ca2+) involvement and timing of pathway activations; all affected ERK activation as early as 3-6min, suggesting simultaneous, not sequential, activation. Therefore, E2 and other estrogenic compounds can produce rapid ERK phosphorylations via nongenomic pathways, using more than one pathway for signal generation.
KW - Ca
KW - EGF receptor
KW - Membrane estrogen receptor
KW - Non-genomic
KW - PI3K
KW - Signaling pathway inhibitors
KW - Src kinase
UR - http://www.scopus.com/inward/record.url?scp=1842638505&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=1842638505&partnerID=8YFLogxK
U2 - 10.1016/j.steroids.2003.12.003
DO - 10.1016/j.steroids.2003.12.003
M3 - Article
C2 - 15072920
AN - SCOPUS:1842638505
SN - 0039-128X
VL - 69
SP - 181
EP - 192
JO - Steroids
JF - Steroids
IS - 3
ER -