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Regulation of Estrogen related receptor alpha localization and signaling by the kinesin KIF17
Nilsa M. Méndez1 and Geri E. Kreitzer2
1Industrial Biotechnology Department, University of Puerto Rico and 2Developmental and Cell Biology, Weill Cornell Medical College
ACT
Abstract
Abstract
Kinesins are motor proteins that transport a variety of cargos such
dehyde 2% and permeabilized with -20oC methanol for 15 secs. Endo- ESRRA was
stained using anti human ESRRA mouse monoclonal primary antibody and Cy5 anti
as organelles, vesicles, RNA, protein complexes and even viruses, to specific
N
destinations in the cell in a microtubule and ATP dependent manner. KIF17
CREM
mouse secondary antibody. DAPI was used as a nuclear stain.
MT
the pictures and they were analyzed using MetaMorph version 6.3r1.
Medium (Fatty Acid Free BSA) for 36 hours at 37oC and 5% CO2. Cells were treated
ESRRA is an orphan
with 100 ng/mL EGF for the final 30 and 60 minutes of the incubation.
nuclear receptor structurally and functionally similar to the classic estrogen
estrogen or estradiol. However, phosphorylation of ESRRA by EGF signaling
pathway can make ESRRA to change its conformation and enhance DNAbinding. ESRRA activity is regulated in part by direct competition with ERs
Figure 3. KIF17 binds to ACT and transports it into the nucleus, were
Phase
contrast
cells . This is a novel regulatory function of KIF17.
Endogenous
ESRRA
MCF-7 (ER positive) cells starts in the nucleus and after the treatment, the
distribution becomes both nuclear and cytoplasmic, while MDA-MB-231 is mostly
altering ESRRA intracellular localization. Overall, a complete understanding of the
MCF-7 cells  ER (+)
activates transcription of CREM dependent genes in murine male germ
ESRRA is mostly nuclear (Figure 4) . Comparing both cell lines, ESRRA localization in
nuclear throughout the whole treatment. Our findings suggest that EGF might be
Results
Results
receptors (ER) but its activity is independent of any known ligands, like
in the outer periphery of the nucleus. After 60 minutes of treatment, ESRRA is
localized within the nucleus and the cytoplasm in MCF-7 cells. In MDA-MB-231 ,
EGF treatment―MCF-7 and MDA-MB-231 cells were starved in DMEM Serum Free
hybrid assay and immunoprecipitation - that one of the cargos that interacts
Endogenous ESRRA (MCF-7 control) is mainly localized in the nucleus,
however after 30 minutes of treating the cells with EGF, ESRRA begins accumulating
Fluorescence microscopy―Nikon Eclipse TE2000-U microscope was used to take
is part of the kinesin family and it was found - by the methods of yeast two-
with is Estrogen related receptor alpha (ESRRA).
Summary
Summary
mechanism by which EGF and KIF17 are involved in the subcellular translocation of
DAPI
ESRRA is essential and may foster the development of new treatments for breast
cancer patients.
genes. It has been previously reported that KIF17 is involved in regulating
T0
(control)
and it must go to the nucleus in order to activate transcription of its target
ESRRA
CREM mediated transcription by interacting with and controlling the
What’s next?
germ cells. Under the light of this precedent, we hypothesize that KIF17
might be involved in regulating nuclear transport of ESRRA, and hence, its
N
MT
activity. Our goal is to reveal the functional significance of KIF17-ESRRA
interaction in breast cancer cells and we will do this by first, determining the
mechanisms by which KIF17 controls the intracellular distribution of ESRRA
T30
(30 mins, +EGF)
intracellular localization of the transcriptional activator ACT in murine male
Measure nuclear:
cytoplasmic ratio
to see if there’s a
significant change
in ESRRA
distribution after
EGF treatment
fluorescence
microscopy.
Knowing
the
mechanisms underlying ESRRA regulation is important because its activity is
related to aggressive tumor behavior and poor prognoses in breast cancer
patients.
Introduction
Introduction
Figure 1. Kinesin
structure.
Kinesins
are motor proteins
that transport a
variety of cargos. The
N-term is the motor
domain that binds to
MT, the coiled-coil
domain
regulates
homodimerization
and the C-term is the
cargo binding domain
Figure 2. KIF17
colocalizes with
microtubules.
GFP-KIF17-FL
(red) and
microtubules
(green)
into the nucleus. ESRRA is an orphan nuclear receptor that is constitutively
active and does not bind to any known physiological ligand. Even though
ESRRA is widely expressed in normal tissues, studies have shown that
ESRRA is an unfavorable biomarker for breast cancer.
Goals
We want to determine the mechanism by which KIF17:
Figure 5. Intracellular distribution of endogenous ESRRA in MCF-7 cells
after EGF treatment
MDA-MB-231 cells  ER (-)
Phase
contrast
1. controls the intracellular distribution of
ESRRA (nuclear vs cytoplasmic)
2. can modulate transcription of ESRRA target
genes
3. regulates interactions of transcriptional coactivators with ESRRA
Materials
Materials and
and methods
methods
Cell lines―the cells used were MCF-7 and MDA-MB-231. These are mammary
epithelial cells Estrogen receptor positive and negative, respectively.
Cell culture―all cells were grown in 10 cm dishes with DMEM medium
supplemented with 10% Fetal Bovine Serum (10% FBS) and 20mM HEPES,
incubated at 37oC and 5% CO2.
Fixation and immunocytochemistry― all cells were rinsed with Phosphate
buffered saline (PBS) with Ca2+ and Mg2+, fixed during 2 minutes in paraformal-
Endogenous
ESRRA
DAPI
T0
(control)
and
Figure 7. Over expression of GFP-ESRRA FL and RFP-KIF17 mut
GE in MCF-7 cells
References
Massarweh, S. and Schiff, R. Resistance to endocrine therapy in breast cancer:
exploiting estrogen receptor/growth factor signaling crosstalk. Endocrine-Related
Cancer (2006) 13, S15-S24
Ciguere, V. and Barry, J. Epidermal Growth Factor-induced signaling in breast
cancer cells results in selective target gene activation by orphan nuclear receptor
T60
(60 mins, +EGF)
immunocytochemistry
Figure 4. KIF17 might regulate ESRRA activity by controlling its transport
T30
(30 mins, +EGF)
ESRRA localization and determine the nuclear:cytoplasmic ratio by
T60
(60 mins, +EGF)
(nuclear vs cytoplasmic) in presence of EGF. We will measure alterations of
Look at up/down
regulation of
transcription of
ESRRA target
genes (+/- EGF, +/KIF17)
Do live, time-lapse
imaging of the
movement of
ESRRA into the
nucleus after EGF
treatment (+/KIF17 GE or tail
domain)
Estrogen-Related α. Cancer Res 2005; 65: (14) 6120-6129
Kotaja, N., Macho, B. and Sassone-Corsi, P. Microtubule-independent and Protein
Kinase A-mediated function of KIF17b controls the intracellular transport of
activator of CREM in testis (ACT). J. Biol. Chem. 280, 31739-31745
Stein, R.A. and McDonnell, D.P. Estrogen-related receptor α as a therapeutic target
Figure 6. Intracellular distribution of endogenous ESRRA in
MDA-MB-231 cells after EGF treatment
in cancer. Endocrine-Related Cancer (2006) 13 S25-S32