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Microsoft word - 20060101_estradiol increases beta-catenin binding.doc

Estradiol Increases Beta-Catenin Binding to Nuclear TCF/LEF: A Novel Hormone-Dependent
Mechanism for Wnt Signaling in the Rat Uteru
Meryl Twarog and Virginia Rider
Department of Biology, Pittsburg State University, Pittsburg, Kansas 66762
An Abstract of a Presentation to: K-INBRE Research Symposium, KSU, Manhattan, KS (January 2006)
Previous studies in our laboratory showed that progesterone pre-treatment of ovariectomized rat uteri
followed by an injection of estradiol increase the number of synchronously proliferating stromal cells five-
fold over that in pregnant rats. Estradiol stimulates the entry of progesterone primed uterine stromal cells
into the cell cycle. In this study, I investigate a mechanism that may underlie the hormonal control of
uterine stromal cell proliferation and differentiation. Specifically, I am analyzing if estradiol stimulates the
nuclear localization of beta-catenin, an important component in the wingless (Wnt) signaling pathway.
Previous studies in the laboratory showed that beta-catenin accumulates in the stromal cell cytoplasm in
progesterone-pretreated rats. However, estradiol was necessary for nuclear translocation of beta-
catenin. Nuclear beta-catenin is known to bind to T cell SEQ CHAPTER \h \r 1factor/lymphoid enhancer
factor (TCF/LEF) at the promoters of Wnt dependent genes and activate transcription. We hypothesized
that estradiol may be required for nuclear localization of beta-catenin and Wnt dependent gene
transcription. Sexually mature ovariectomized Sprague Dawley rats were injected subcutaneously (s.c)
with progesterone (2 mg) for three consecutive days. Uteri were removed under anesthesia from this
group (0 hE) and stored without further treatment at –80 C. A second group of progesterone pre-treated
rats were injected s.c. with estradiol (0.2 µg) on day 4 and the uteri were removed 6 h later (6 hE). Uterine
horns from both samples were homogenized in 5 volumes of homogenization buffer (0.3 M sucrose, 10
mM Tris-HCl, pH 7.5, 3 mM Mg acetate, 1 mM dithiothreitol) containing protease inhibitors. An agarose-
conjugated beta-catenin antibody (Santa Cruz, sc1496) was added to uterine extracts and samples were
kept on a nutator for 12 h at 4 C. The agarose beads were washed three times in PBS-0.1 M NaCl.
Denaturing sample buffer containing sucrose dye was added and the samples were heated for 5 min at
95 C. The immunoprecipitates and Jurkat cell lysates (20 microliters, positive control, Upstate) were size
fractionated by SDS-PAGE and transferred to nitrocellulose membranes. The nitrocellulose membranes
were reacted with a LEF antibody (2 mg/ml, Upstate). Some samples were treated identically except the
membranes were reacted without the LEF primary antibody (negative control). The blots were washed
and reacted with a goat anti-mouse HRP-conjugated secondary antibody (1:25,000, Pierce). Proteins
were detected by chemiluminescence using the SuperSignal West Femto Maximum Sensitivity Substrate
Kit (Pierce). A protein that migrated with a mass of approximately 60 kDa was detected in the
immunoprecipitates from progesterone-pretreated uteri (0 hE), those injected with estradiol (6 hE) and
Jurkat cell extracts (positive control). The amount of this protein was greater in the immuoprecipitates
from uterine extracts of rats treated with estradiol compared with extracts from progesterone-pretreated
uteri. The size of the major reactive protein is consistent with that reported for mouse TCF/LEF (~ 57
kDa). These results, when coupled with our previous localization studies, support a role for estradiol in
the translocation of beta-catenin from the cytoplasm of progesterone pretreated stromal cells. The results
are consistent with the idea that progesterone initiates Wnt signaling but estradiol is necessary for beta-
catenin localization to the nucleus and the entry of stromal cells into the cell cycle.



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