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Mechanisms for regulating the expression of the TATA-binding protein
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Mechanisms for regulating the expression of the TATA-binding protein
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Content
MECHANISMS FOR REGULATING THE EXPRESSION OF THE
TATA-BINDING PROTEIN
by
Jody Arlene Fromm
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)
August 2007
Copyright 2007 Jody Arlene Fromm
Dedication
To my family.
ii
Acknowledgments
The support of several important individuals made completion of this work
possible. I’d like to first acknowledge my PI, Dr. Debbie Johnson, whose guidance has
been invaluable. I am forever grateful for the training I received with Debbie as my
mentor. Debbie’s support has carried me through many difficult professional and
personal times with grace. She has also been my pseudo-mom when I needed it the
most.
My committee members were carefully picked based on their creative research
projects and their sincerity for the training of PhD students. Dr. Baruch Frenkel has
always attended my yearly committee meetings with a sincere intention to propose ideas
to make my project of significant impact. He was always excited about the progress of
my project and was very supportive of its success. Dr. Judy Garner has been a true gem
throughout my most frustrating troubleshooting times. She always found time for
discussions and provided many protocols and reagents. I appreciate all of the time and
input all of my committee members have contributed. I never once felt like my project
was not one of their own.
Dr. Michael Stallcup has been a significant supporter of my career path. He
supplied several very strong letters of recommendation for me in my post-doc search,
despite his demanding schedule. I am very appreciative of the time he has spent on my
behalf and his advice.
iii
Dr. Joeseph Hacia has been a valuable mentor and friend to me. His support and
encouragement has been infinite. He has let me vent, panic, and rejoice without
judgement. He has a true passion for science and teaching that I admire.
I have some of the greatest friends a person could ask for. They have gone
above and beyond to offer their support. Aimee, Karrie, Chastity, Karen, Nicole,
Tessa, and Ali, I am blessed to have you in my life and your love is unforgettable.
My lab mate and friend, Annette Woiwode, has made many Saturdays, Sundays,
and midnight hours in the lab appear ‘fun’ despite the stress associated with the
long hours.
My parents have always offered encouragement and support even when
they didn’t understand my intentions. They have given me all the qualities that I
possess that have played a large role in my achievements. For that, I am eternally
indebted. I am so lucky to have siblings, Jenny, Benson, and Jackie, that want
nothing but the best for each other and would give up their last dollar to any one
of us. Our parents should be proud of creating a dynamic most families only
dream of.
Star deserves the biggest “thank you”. He received the brunt of my
frustrations associated with attaining this degree. With my family and friends
being so far away, his presence and support was often times a blessing. He
believed in me and was always very vocal of his support, and for that, I am
grateful. He rarely complained about the long hours lab required, lessening the
stresses involved with trying to balance school and personal life. He, along with
iv
my friends and family, are as excited as I am for the completion of this
dissertation.
v
Table of Contents
Dedication ii
Acknowledgments iii
List of Figures ix
Abstract x
CHAPTER 1: Introduction 1
1.1 TATA-BINDING PROTEIN 1
1.2 RAS MEDIATED SIGNAL TRANSDUCTION 2
1.3 EPIDERMAL GROWTH FACTOR RECEPTOR FAMILY 3
1.4 TBP, EGFRs, AND CANCER
5
CHAPTER 2: Analysis of TBP regulation by EGFR1, EGFRvIII, and HER2 8
2.1 RESULTS 9
2.1.1. Key cell lines have select expression of EGFR1, EGFRvIII, or HER2 9
2.2.2 EGFR1 and EGFRvIII, but not HER2, enhance the expression of
TBP 11
2.3.3 EGFR1 and EGFRvIII but not HER2 transcriptionally regulate TBP 15
2.2 SUMMARY 19
2.3 MATERIALS AND METHODS 20
2.3.1 Cell Culture 20
2.3.2 Western Blot Analysis 21
2.3.3 Plasmid DNAs 22
2.3.4 Transient Transfections 23
2.3.5 Preparation of Cell lysates and Luciferase Assay 24
CHAPTER 3: Transcriptional regulation of TBP by EGFR1 25
3.1 RESULTS 26
3.1.1 EGFR1 transcriptionally regulates TBP via a putative ETS site 26
3.1.2 Transcriptional regulation of TBP by EGFR1 requires Ras-MAPK 29
3.1.3 Elk-1 is targeted to the TBP promoter by EGFR1 33
3.2 SUMMARY 40
3.3 MATERIALS AND METHODS 40
3.3.1 Cell Culture 40
vi
3.3.2 Western Blot Analysis 41
3.3.3 Plasmid DNAs 41
3.3.4 Transient Transfections 42
3.3.5 Preparation of Cell lysates for Protein, Luciferase and β-gal Assays 43
3.3.6 Site-directed mutagenesis 43
3.3.7 Real time PCR 44
3.3.8 Chromatin Immunoprecipitation Assay 45
CHAPTER 4: Transcriptional regulation of TBP by EGFRvIII 46
4.1 RESULTS 46
4.1.1 EGFRvIII does not require the Elk-1 response element 46
4.1.2 EGFRvIII retains responsiveness with a -54/-1 TBP minimal
promoter 47
4.1.3 EGFRvIII requires a putative AP-1 response element to
transcriptional regulate TBP promoter 52
4.1.4 EGFRvIII directly targets c-Jun and c-Fos to the TBP promoter 52
4.2 SUMMARY 55
4.3 MATERIALS AND METHODS 55
4.3.1 Cell Culture 55
4.3.2 Western Blot Analysis 56
4.3.3 lasmid DNAs 56
4.3.4 Transient Transfections 56
4.3.5 Preparation of Cell lysates for Protein, Luciferase and β-gal Assays 57
4.3.6 Site-directed mutagenesis 57
4.3.7 Real time PCR 57
4.3.8 Chromatin Immunoprecipitation Assay 58
CHAPTER 5: Internalization distinguishes EGFR1 and EGFRvIII signaling 59
5.1 RESULTS 59
5.1.1 Overexpression of c-Jun and c-Fos regulates TBP promoter activity 59
5.1.2 The rate of EGFR1 internalization influences the activation of
downstream signaling cascades 60
5.2 SUMMARY 64
5.3 MATERIALS AND METHODS 64
5.3.1 Cell Culture 64
5.3.2 Western Blot Analysis 64
5.3.3 Plasmid DNAs 65
5.3.4 Transient Transfections 65
vii
5.3.5 Preparation of Cell lysates for Protein, Luciferase Assays 65
CHAPTER 6: Discussion 66
6.1 Distinguishing the distinct gene targets of the EGFRs is of
biological relevance 66
6.2 EGFR1, but not HER2, regulates TBP gene expression 67
6.3 EGFR1 requires Elk-1 to regulate TBP gene expression 68
6.4 JNK1 positively regulates, whereas JNK2 negatively regulates the
phosphorylation state of Elk-1 68
6.5 EGFR1 and EGFRvIII differentially regulate TBP promoter activity 70
6.6 A composite ETS/AP-1 site within the TBP promoter allows for differential
regulation by EGFR1 and EGFRvIII 71
6.7 Endocytosis plays a key role in distinguishing EGFR1 and EGFRvIII
mediated signal transduction 72
References 74
viii
List of Figures
Figure 1: Characterization of cell lines. 12
Figure 2: TBP protein levels are increased in EGFR1 and EGFRvIII cell lines. 13
Figure 3: EGFR1 transcriptionally regulates TBP. 16
Figure 4: EGFRvIII transcriptionally regulates TBP. 17
Figure 5: HER2 does not transcriptionally regulate TBP. 19
Figure 6: EGFR1 targets an ETS response element. 27
Figure 7: EGFR1 requires sequences within -54/-1 TBP promoter fragment 29
Figure 8: EGFR1 requires MAPKs to transcriptionally regulate TBP 30
Figure 9: TBP expression is differentially regulated by the JNKs. 32
Figure 10: Elk-1 regulates TBP gene expression. 35
Figure 11: Elk-1 directly modulates EGF-induced TBP promoter and is
differentially regulated by JNK1 and JNK2.
36
Figure 12: EGFR1 does not target an adjacent AP-1 site. 38
Figure 13: EGFRvIII transcriptionally regulates TBP via a non-ETS response
element.
48
Figure 14: EGFRvIII requires sequences within -54/-1 TBP promoter fragment. 50
Figure 15: EGFRvIII regulates TBP via an AP-1 response element 52
Figure 16: Activated AP-1 complexes can regulate the TBP promoter. 60
Figure 17: Inhibition of EGFR1 internalization mimics EGFRvIII signaling. 62
ix
Abstract
The Epidermal Growth Factor Receptor (EGFR) family regulates essential
biological processes upon receptor activation and deregulation of these receptors is
detrimental to cellular homeostasis. HER2 and EGFR1 are two members of the EGFR
receptor tyrosine kinase family. Several human cancers are linked to either HER2 or
EGFR1 overexpression or expression of EGFR1 variants, like EGFRvIII. One important
gene target of EGF-mediated signaling is the TATA-binding protein (TBP) (64). TBP is a
central eukaryotic transcription initiation factor required by all nuclear RNA polymerases
for transcription. Selective changes in gene expression patterns occur when TBP
expression levels are altered (8, 57) and increased expression of TBP has been shown to
promote cellular transformation (28). This study focuses on if and how activated HER2,
EGFR1, and EGFRvIII regulate TBP expression. Here we show that EGFR1 and
EGFRvIII, but not HER2, transcriptionally regulate TBP expression. EGFR1 regulates
the TBP promoter via the recruitment of Elk-1 and EGFRvIII regulates the TBP promoter
via recruitment of c-Jun and c-Fos. Furthermore, EGFR1 and EGFRvIII-mediated
regulation of TBP expression is differentiated by transient versus sustained receptor
activation, which is an effect of differential receptor internalization and recycling rates.
Aside from the significance associated with understanding the mechanism by which this
integral transcription factor is regulated in a transformed environment, the identity of
specific downstream targets distinguishing the EGFRs is of clinical significance.
x
CHAPTER 1: Introduction
1.1 TATA-BINDING PROTEIN
The TATA-binding protein (TBP) is a ubiquitously expressed
indispensable eukaryotic transcription initiation factor. TBP binds TATAa/tAa/t
consensus sequences in TATA-box containing promoters to initiate transcription,
but for TATA-less containing promoters, TBP recruitment relies on specific
protein-protein interactions. Unique protein-protein and protein-DNA interactions
designate TBP for participation in RNA polymerase I, RNA polymerase II, or
RNA polymerase (pol) III dependent transcription (24). TBP can be a limiting
factor for RNA pol I (57, 64) and RNA pol III-dependent transcription (53, 55, 56,
64). Small increases in TBP expression result in increased rRNA and tRNA
production, which is a hallmark of cancer cells. Most RNA pol II promoters are
unaffected by alterations in TBP expression levels but a select few are either
increased or decreased depending on the promoter structure and regulatory
elements (8, 38, 48). TBP expression levels, therefore, have the potential to
disrupt cellular homeostasis. The effects of tumor suppressors and oncogenic
signaling on TBP expression or function have differential effects depending on the
cell type and cellular context. We have shown that modest increases in the
expression of TBP promotes cellular transformation (28, 29) and TBP has been
shown to be a direct target of the tumor suppressor, p53. p53 can specifically
inhibit RNA pol III activity by sequestering TBP to prevent the formation of
functional TFIIIB complexes (9). Oncogenic Ras results in increased TBP gene
1
expression. Exogenous two-fold increases in TBP expression increases cellular
proliferation rates in mouse embryo fibroblast (MEF) cells, and in Rat1A cells, it
promotes anchorage-independent growth without changing their proliferation
rates (30). The in vivo consequences of increased TBP expression in Rat1A cells
also enhances tumor formation in nude mice. Nude mice subcutaneously injected
with these Rat1A cells containing only a 30% increase in TBP expression form
significantly larger tumors than those mice injected with Rat1A-vector cells (28).
Deregulation of TBP expression has biological consequences as well, as TBP
expression levels have been shown to be increased in human colon tumor tissue
compared to matched normal tissue (28). Collectively, this work supports that
TBP expression is tightly regulated and certain oncogenic pathways induce TBP
expression to promote transformation.
1.2 RAS MEDIATED SIGNAL TRANSDUCTION
Transcription of TBP can be increased via upstream activators of Ras,
such as, phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) mediated
activation of protein kinase C (PKC) (19, 20), and the hepatitis B virus (HBV) X
protein (57). Expression of a oncogenic form of Ras also induces TBP gene
expression (55) and PKC and HBV-X mediated signaling induces the TBP
promoter through the activated Ras. Ras proteins are small guanine triphosphate
kinases (GTPases) that provide a critical link between the transduction of
extracellular cues and the regulation of cellular proliferation and differentiation.
Activation of growth factor receptor tyrosine kinases by extracellular molecules
2
results in a guanine diphosphate kinase (GDP) to GTP switch of Ras proteins
leading to a signaling cascade comprised of several serine/threonine kinases and
nuclear transcription factors. Ras can directly interact with at least three effector
molecules, Raf, RalGDS, and PI3K. Raf and RalGDS are upstream activators of
MEK and MAPK that serve to activate specific transcription factors, such as Ets
and AP-1, to regulate translation and proliferation. PI3K activates AKT, also
known as protein kinase B (PKB), which regulates several molecules, such as,
Myc, mTOR, and GSK3, to regulate cell survival and cell cycle progression.
Deregulation of these signaling pathways can lead to cellular transformation,
therefore, it is important to define the key upstream and downstream components
of these pathways. It is well known that many ligand-activated receptor tyrosine
kinases (RTK) are upstream activators of Ras. One RTK family of particular
interest due to their prevalence in cancer is the EGFR family.
1.3 EPIDERMAL GROWTH FACTOR RECEPTOR FAMILY
We have discovered that epidermal growth factor (EGF)-mediated
activation of EGFR1 regulates the expression of TBP via Ras-MAPK signaling
(64). The EGFR family is comprised of four transmembrane receptor tyrosine
kinases (RTKs), namely, EGFR1 (ErbB1/HER1), HER2 (ErbB2), HER3 (ErbB3),
and HER4 (ErbB4). These receptors share a similar kinase domain structure and
homology but differ in their extracellular domains and carboxy terminal tails (49).
The EGFR family plays a vital role during development and is fundamental in
regulating cellular proliferation, cell survival, and cell migration. EGFR1 and
3
HER2 signal through Ras-MAPK and PI3K to regulate essential biological
processes depending on the cellular context. HER2 is a strong activator of PI3K/
AKT and regulator of cell survival and cell migration whereas EGFR1 is a strong
activator of Ras-MAPK and regulator of cellular proliferation and cytoskeletal
reorganization. The complexity of the signal transduction pathways activated by
these receptors makes it challenging to define their specific gene targets. EGFR1
and HER2 amplification and/or overexpression is prevalent in a variety of human
cancers. EGFR1 overexpression is especially common in gliomas and HER2
overexpression is especially common in breast cancer. In most cancers,
overexpression of HER2 and EGFR1 results from gene amplification, and unique
to EGFR1, this can result in the formation of a variety of genetic mutant forms of
EGFR1. The tumor specific receptor, EGFRvIII, is the most common genetic
variant form of EGFR1 distinguished by an in-frame deletion of exons 2-7,
corresponding to amino acids 6-273, in the extracellular domain. The EGFRvIII-
specific deletion results in a novel extracellular domain architecture that mimics
an activated receptor that is unable to bind EGF.
Tumors with aberrant expression of EGFR1, EGFRvIII, or HER2 are
clinically challenging to treat due to reoccurring drug resistance. A more
promising clinical approach in the treatment of gliomas, for example, is to
combine chemotherapy with specific inhibitors of small molecules (41). For this
reason, it is imperative to identify the specific intracellular downstream molecules
specific for EGFR1, EGFRvIII, and HER2-mediated transformation. Studying
4
these mechanisms, however, are complicated by various factors: 1) signaling
events are sensitive to the level of EGFR1 expressed; 2) the presence of other
EGFRs may obscure the signaling events exclusively associated with EGFR1,
EGFRvIII or HER2; 3) biological responses to mitogens and cytokines are often
cell type specific; 4) prolonged versus transient signals may differentially regulate
cellular responses (37). These issues have been considered as we set out to define
specific signaling events mediated by these receptors that regulate TBP
expression.
1.4 TBP, EGFRs, AND CANCER
Aberrant activators of Ras, and the consequences in gene expression that
follow, serve to promote cellular transformation. Deregulation of Ras can occur
via amplification of EGFRs, overexpression of RTK ligands and cytokines, or
mutations in proteins that negatively control Ras activation. The oncogenic
potential of Ras requires increases in TBP gene expression to transform Rat1A
cells (30) and the activation of Ets transcription factors downstream of Ras are
required to transform NIH3T3 cells (30, 58). The Ets family of transcription
factors consists of approximately 30 members, sub-grouped into nine major
groups depending on their structural composition. The Ets subfamilies are ETS,
ERG, ELG, TEL, PEA3, ELF, SP1, ERF, and TCF. Each subfamily contains a
divergent combination of several conserved domains: an Ets DNA binding
domain, an activation domain, a repression domain, an auto-inhibitory domain,
and a pointed domain. The expression profiles of Ets factors are cell type
5
dependent and temporally regulated. Ras enhances the transcription of TBP
through a functional ETS binding site within the TBP promoter (30). This ETS
site overlaps with a consensus binding site for AP-1. Oncogenic Ras can
synergistically regulate promoters that contain composite ETS/ETS and ETS/
AP-1 promoter elements, such as, the collagenase, c-Fos, HB-EGF and junD
genes (17, 18, 35, 39, 59). AP-1 response elements, are bound by either
homodimers of c-Jun or heterodimers of c-Jun and c-Fos family members. The c-
Jun family is comprised of c-Jun, JunB, and JunD. The c-Fos family is comprised
of c-Fos, Fra-1, and Fra-2.
As amplification of EGFR1 precedes EGFRvIII expression and the
aggressive tumor state, it is conceivable that the, in vivo, co-expression of
oncogenic EGFR1 and EGFRvIII allows for powerful regulation of TBP and/or
other genes by exploiting promoters containing composite ETS/AP-1 response
sites. Our work has shown that Ras mediated stimulation of the TBP promoter
requires a responsive ETS site, and we propose that EGFRvIII might enhance
TBP expression and/or other genes by targeting adjacent AP-1 sites. Also shown
here is that not all oncogenic signaling pathways increase TBP expression. Our
findings show several examples of proteins displaying distinct functions despite
possessing many structural and biochemical similarities.
Here we defined unique mechanisms for regulating the TATA-binding
gene. We have characterized the signal transduction pathways that serve to
regulate the transcription of TBP in either a positive or negative manner. We have
6
identified the transcription factors directly responsible for the expression of TBP
in different cellular contexts. This study focused on the transcriptional regulation
of TBP downstream of oncogenic growth factor receptors in the EGFR family.
7
CHAPTER 2: Analysis of TBP regulation by EGFR1, EGFRvIII, and HER2
The potential regulation of TBP expression by EGF was initially addressed
in a mouse embryo fibroblast JB6 cell line. When JB6 cells were stimulated with
EGF an increase in TBP message and protein levels were observed (64).
Regulating the expression levels of proteins can occur through multiple
mechanisms. Genes expression can be regulated by enhanced promoter activity
or transcription of the gene, enhanced stability of the mRNA providing a potential
for enhanced translation, or enhanced stability of the half-life of the protein by
preventing its degradation. The EGF-mediated increase in TBP expression was
determined to be regualted at the transcriptional level, as EGF stimulation of JB6
cells induced TBP promoter activity (64). This study also showed that the
transcriptional regulation of TBP by EGF-activated EGFR1 required the
activation of Ras. These initial studies did not address this regulatory mechanism
in human cells nor did it address whether this was specific for the expression of
EGFR1. Given that HER2 can also signal through Ras it may also be a potential
regulator of TBP expression. We also know that EGFRvIII has the same c-
terminal tail as EGFR1, so it may also be a potential regulator of TBP expression.
The potential of EGFR1, EGFRvIII, and HER2 to regulate TBP expression was
addressed in both human and mouse cell lines in the absence of co-expression of
multiple EGFRs.
8
2.1 RESULTS
2.1.1. Key cell lines have select expression of EGFR1, EGFRvIII, or HER2
To detail the potential regulatory consequences of oncogenic EGFR1,
EGFRvIII, and HER2 expression on TBP expression we used a combination of
cell lines that express only one of these receptors. In order to define the signal
transduction pathways specific for EGFR1, EGFRvIII, and HER2, previously
characterized cell lines were used that individually express EGFR1, EGFRvIII, or
HER2 (for details see Materials and Methods). Immunoblot analysis first
confirmed the expression of these receptors in each cell line. NR6 and NIH3T3
parental cell lines have undetectable levels of EGFR1, EGFRvIII, and HER2
(Figure 1A). Specific expression of HER2, EGFR1, or EGFRvIII was detected
for each respective cell line as shown (Figure 1A). The U87 parental cell line has
undetectable levels of EGFR1, EGFRvIII, and HER2 (Figure 1B, Left). The U87-
EGFR1 stable cell line expresses EGFR1 only and the U87-EGFRvIII cell line
expresses EGFRvIII only (Figure 1B). HER2 expression was detected in the
MCF7-HER2 cell line but not in the MCF7 parental cell line (Figure 1B, Right).
Conditions were then established to activate and inactivate these receptors
as measured by an increase or decrease in receptor tyrosine phosphorylation.
Phospho-tyrosine-specific antibodies were used to confirm the activation status of
EGFR1, EGFRvIII, and HER2 under different conditions. To inhibit receptor
activation cells were treated with the EGFR1 and EGFRvIII-specific chemical
9
AG825 block the activation potential by competing with ATP for binding in the
receptor’s auto-kinase domain. Lysates derived from untreated, AG1478, or FBS
treated NR6-Vector, NR6-EGFR1, and NR6-EGFRvIII cells were
immunoprecipitated with α-EGFR or α-p-tyrosine and immunoblot analysis was
then performed with α-p-tyrosine and α-EGFR. In the NR6-EGFR1 cell line,
FBS treatment induced activation of EGFR1 and AG1478 treatment inhibited
EGFR1 activation (Figure 1C, Left). There was a detectible level of constitutive
auto-tyrosine phosphorylation of EGFR1, which has been previously reported
(Figure 1C Left, Bottom) (26). In the NR6-EGFRvIII cell line, constitutive
activation of EGFRvIII was detected in the absence of serum or stimuli and
AG1478 treatment abrogated EGFRvIII activation (Figure 1C, Left). The NR6-
HER2 cell line showed constitutive activation of HER2 independent of serum
stimulation while AG825 inhibitor treatment abrogated the activation of HER2
(Figure 1C, Right).
Receptor activation was also confirmed in the U87 cell lines by
immunoblot analysis using an antibody that recognizes p-EGFR1 and p-
EGFRvIII. The control cell line had barely detectable levels of p-EGFR1 with
EGF treatment and detection of total EGFR1 in the control and EGFRvIII cell
lines required a significant overexposure of chemiluminescence to film (Figure
1D). The U87-EGFR1 stable cell line displayed EGF induced receptor activation
and also displayed a low level of autophosphorylation, which has been previously
reported (37, 45, 46). The U87-EGFRvIII cell line displayed constitutive
10
activation of EGFRvIII (Figure 1D). AG1478 treatment inhibited receptor
activation of EGFR1 and EGFRvIII (Figure 1D). Constitutive activation of HER2
was detected in the MCF7-HER2 cell line and this activation was abrogated with
the AG825 inhibitor (Figure 1E). Collectively this data confirms that each
specific receptor is appropriately expressed and activated in all cell lines.
2.2.2 EGFR1 and EGFRvIII, but not HER2, enhance the expression of TBP
To test whether the activation of EGFR1, EGFRvIII, or HER2 affected
cellular TBP protein levels, immunoblot analysis was used. NR6-EGFR1 and
NR6-EGFRvIII and NIH3T3-EGFRvIII cells exhibited a significant increase in
cellular protein levels of TBP compared to lysates derived from vector or HER2
expressing cells (Figure 2A, B). Consistent with these results, U87-EGFR1 and
U87-EGFRvIII cells displayed an increase in the cellular levels of TBP protein
compared to lysates derived from U87-vector cells (Figure 2C). Lysates derived
from human MCF7-HER2 cells showed comparable cellular levels of TBP protein
expression to that of the non-HER2 overexpressing, MCF7 cells (Figure 2D).
To confirm that the observed increases in TBP protein levels were a direct
result of EGFR1 or EGFRvIII activation, TBP expression was analyzed in the
presence and absence of AG1478 treatment.
11
EGFRvIII
NR6
HER2
EGFR1
HER2
EGFRvIII
Parental
EGFR1
Vector
EGFRvIII
NIH3T3
HER2
Parental
EGFRvIII
HER2
A.
EGFRvIII
HER2
EGFR1
U87
EGFRvIII
Parental
EGFR1
Parental
HER2
MCF7
HER2
B.
C.
IP: α-pTyr
p-HER2
HER2
p-HER2
FBS:
HER2
Vector
IP: α-HER2
HER2
NR6
-
+ + -
AG825: -
+ + -
D.
EGFR1
EGFRvIII
p-EGFR1
p-EGFRvIII
EGFRvIII
α-pTyr
IB:
ΕGFR
IB:
+ - + - - EGF: +
AG1478:
+
+ + - + - - -
EGFR1
Vector
U87 MCF7-HER2
p-HER2
HER2
AG825: - +
E.
EGFR1
Vector
EGFRvIII
NR6
FBS:
+ -
AG1478:
+ -
+
- - -
+
-
+
- - -
p-EGFRvIII
EGFR1
EGFRvIII
p-EGFRvIII
p-EGFR1
EGFR1
EGFRvIII
AG1478: - - + +
-
IP: α-pTyr:
p-EGFR1
IP: α-EGFR1:
+
FBS: + + + + + +
Figure 1. Characterization of cell lines used. A, B). Immunoblot analysis was performed using
whole cell lysates (WCL) derived from logarithmically growing cells using EGFR1 or HER2
specific antibodies. C). Immunoprecipitation (IP) was performed using the designated antibodies
with 400 ug of WCL derived from serum starved NR6 cells treated overnight with FBS (10%),
AG1478 (10 uM), or EGF (50ng/ml). Immunoblot analysis was performed after IP using pTyr,
EGFR1, or HER2 antibodies (left panel). Immunoblot analysis was performed with pTyr or HER2
antibodies after IP of 400 ug of WCL derived from NR6 cells treated with FBS (10%) or AG825
(25uM) overnight (right panel). D). Immunoblot analysis using pEGFR1 or EGFR1 antibodies
was performed on lysates derived from U87 cells treated with EGF or AG1478 overnight. E).
Immunoblot analysis was performed on IP WCL derived from MCF-HER2 stable cell lines treated
with/without AG825 overnight. HER2 antibodies were used for the IP and pTyr antibodies or
HER2 antibodies were used for the immunoblot analysis.
Lysates derived from the AG1478 treated NR6 and U87 cells expressing
EGFR1 or EGFRvIII displayed TBP protein levels comparable to that observed
for the vector untreated cells (Figure 2E, F). Lysates derived from NR6-vector
and NR6-HER2 cell lines showed no change in TBP protein levels independent of
12
receptor activation state (Figure 2F). Similarly, the HER2 inhibitor also had no
effect on TBP protein expression in MCF7-HER2 cells (Figure 2G). This data
confirms that EGFR1 and EGFRvIII positively regulate the expression of TBP but
HER2 does not.
TBP
β-actin
Parental
NIH3T3
EGFRvIII
HER2
0
1
2
3
4
5
6
7
Parental
EGFRvIII
HER2
Fold change in TBP
protein levels
B.
0
1
2
3
4
Vector
EGFR1
EGFRvIII
HER2
Fold change in TBP
protein levels
β-actin
Vector
EGFR1
EGFRvIII
HER2
TBP
NR6
A.
Vector
EGFR1
EGFRvIII
Fold change in TBP
protein levels
Vector
EGFR1
EGFRvIII
U87
TBP
β-actin
C. D.
HER2
TBP
β
-actin
Parental
MCF7
0
1
2
3
4
HER2
Parental
Fold change in TBP
protein levels
0
10
8
2
4
Figure 2. TBP protein levels are increased in EGFR1 and EGFRvIII stable cell lines. A-D).
Immunoblot analysis was performed using WCL derived from logarithmically growing cells
using antibodies for TBP and β-actin.
13
F.
AG825: - + - +
NR6
TBP
β-actin
- - - - - -
- - - -
EGFR1
Vector
EGFRvIII
AG1478:
+
-
+
-
+
-
Vector
HER2
E.
Parental
EGFR1
EGFRvIII
U87
TBP
β-actin
EGFR1
Vector
EGFRvIII
AG1478: +
-
+
-
+
-
G.
HER2
EGFR1
Vector
EGFRvIII
HER2
TBP protein levels
0
1
2
3
4
Fold change in
AG825: - + - - - - - -
- - AG1478:
+
-
+
-
+
-
TBP protein levels
0
1
2
3
Fold change in
AG1478: -
+
- - + +
0.0
0.5
1.0
1.5
TBP protein levels
Fold change in
AG825: - +
MCF7-HER2
AG825:
TBP
β-actin
- +
Figure 2. TBP protein levels are increased in EGFR1 and EGFRvIII stable cell lines. E-G).
Immunoblot analysis was performed on lysates derived from stable cell lines treated with AG1478
(10uM) or AG825 (25uM) or DMSO overnight. The films were quantified and the fold changes in
TBP protein levels were normalized to endogenous β-actin levels and graphed. Immunoblot
analysis was performed using antibodies against TBP and β-Actin. The fold change in TBP
protein levels are expressed as the mean ± SEM based on at least three independent
determinations.
14
2.3.3 EGFR1 and EGFRvIII but not HER2 transcriptionally regulate TBP
Previous work showed that oncogenic Ras transcriptionally regulates TBP,
so we addressed whether EGFR1, EGFRvIII, or HER2 transcriptionally regulate
TBP. In NR6 and U87 cells, exogenous expression of EGFR1 induced a
transiently expressed TBP promoter reporter construct in an AG1478 sensitive
manner (Figure 3A). EGFR mediated transcriptional regulation of TBP was also
analyzed in the U87-EGFR1 and NR6-EGFR1 stable cell lines. In both the NR6-
EGFR1 and U87-EGFR1 stable cell lines TBP promoter activity was also
regulated in an AG1478 sensitive manner (Figure 3B). As expected, the human c-
Fos promoter activity was also regulated through EGFR1; meanwhile, activity of
the human B3-Integrin promoter activity remained unaffected upon AG1478
treatment (Figure 3B).
Despite the homology between EGFRvIII and EGFR1, previous studies
propose that they are functionally different (23, 40, 45, 50). We addressed the
potential transcriptional regulation of the TBP promoter by EGFRvIII by
transiently expressing EGFRvIII in the U87 or NR6 parental cell lines. Transient
expression of EGFRvIII induced TBP promoter activity and this induction was
inhibited upon AG1478 treatment (Figure 4A). In U87-EGFRvIII and NR6-
EGFRvIII stable cell lines, TBP promoter activity was also AG1478 sensitive
(Figure 4B). The human c-Fos promoter was also repressed upon inhibitor
treatment while the CMV promoter activity remained unaffected (Figure 4B).
15
TBP-Luc
c-Fos-Luc
B3-Integrin-Luc
B.
A.
AG1478: - + - +
0
2
3
4
5
6
7
1
EGFR1:
+ + - -
promoter activity
Fold change in TBP
NR6
U87
promoter activity
EGFR1: + -
Fold change in TBP
8
4
2
0
EGF:
+ + + +
EGF:
+ +
NR6-EGFR1
0.0
0.5
1.0
1.5
AG1478: - +
promoter activity
Fold change in TBP
EGF:
+ +
U87-EGFR1
0.0
0.5
1.0
1.5
AG1478: - +
promoter activity
Fold change in
- + - +
EGF: + + + + + +
Figure 3. EGFR1 transcriptionally regulates TBP. A). Cells were transiently co-transfected with
4 ug of an EGFR1 expression vector and 2 ug of a promoter reporter construct. Cells were serum
starved the following day and 6-8 hours later treated with EGF (50 ng/ml) and/or 2 uM (NR6) or
10 uM (U87) AG1478 overnight. Cells were harvested the following day and analyzed for
luciferase activity. Fold changes were calculated based on control (TBP promoter + empty
expression vector), normalized to total protein. The values shown are the mean ± SEM of at least
3 independent determinations. B). Cells were transfected with the promoter reporter construct
and serum starved the following day. 6-8 hours post-serum starvation, cells were treated with
EGF (50 ng/ml) and/or 2 uM (NR6) or 10 uM (U87) AG1478 overnight. Cells were harvested the
following day and analyzed for luciferase activity and normalized to total protein. The data
represents at least 3 independent experiments.
16
TBP-Luc
c-Fos-Luc
CMV-Luc
B.
AG1478:
0.0
0.5
1.0
1.5
-
+
- + - +
U87-EGFRvIII
promoter activity
Fold change in
NR6-EGFRvIII
0.0
0.5
1.0
1.5
AG1478: - +
promoter activity
Fold change in
NIH3T3-EGFRvIII
0.0
0.5
1.0
1.5
AG1478: -
+
- +
promoter activity
Fold change in
NIH3T3
0.0
0.5
1.0
1.5
AG1478: -
+
- +
promoter activity
Fold change in
C.
A.
U87
8
4
2
0
EGFRvIII: - +
promoter activity
Fold change in TBP
NR6
AG1478: - + - +
0
2
3
4
5
1
EGFRvIII:
+ + - -
promoter activity
Fold change in TBP
Figure 4. EGFRvIII transcriptionally regulates TBP. A). Cells were transiently co-transfected
with 4 ug of an EGFRvIII expression vector and 2 ug of the designated promoter reporter
construct. Cells were serum starved the following day and 6-8 hours later treated with AG1478, 2
uM (NR6) or10 uM (U87), overnight. Cells were harvested the following day and analyzed for
luciferase activity. Fold changes were calculated based on control (TBP promoter + empty
expression vector), normalized to total protein. B, C). Cells were transfected with the promoter
reporter construct and serum starved the following day. Cells were treated and isolated as
described in “A”. The values shown are the mean ± SEM of at least 3 independent determinations.
17
This was confirmed in an additional cell, NIH3T3-EGFRvIII, where AG1478
treatment inhibited TBP promoter activity while having little effect on CMV
promoter (Figure 4C, Left). As expected, treatment of NIH3T3 parental cells with
AG1478 had no effect on TBP or CMV promoter activity (Figure 4C, Right).
Collectively, this data indicates that both EGFR1 and EGFRvIII transcriptionally
regulate TBP expression. While HER2 overexpression did not affect TBP protein
levels, (Figure 3C) we addressed whether HER2 might affect TBP promoter
activity. Through an unknown mechanism, HER2 becomes constitutively active
once a specific threshold of cell surface HER2 concentration is reached. The
level of exogenous HER2 expression was varied in transient transfection assays to
establish constitutive activation of the receptor as determined by spontaneous
induction of the human c-Fos promoter (data not shown, Figure 5A). Under these
various conditions where constitutive activation of exogenously expressed HER2
activated the c-Fos promoter, the TBP promoter or a non-inducible human c-Fos
promoter mutant were not activated (Figure 5A). The effect of stable expression
of constitutively active HER2 on TBP promoter activity was addressed in the NR6
and MCF7 cell lines. TBP promoter activity in the NR6-HER2 cell line was
refractory to treatment with AG825 (Figure 5B). Similarly, in the MCF-HER2
stable cell line TBP promoter activity was unaffected by AG825 treatment while a
decrease in c-Fos promoter activity was observed (Figure 5B, Right). These
results confirm that HER2 activation does not regulate TBP promoter activity.
18
A.
TBP-Luc
c-Fos-Luc
c-Fos-mutant-Luc
NR6
0
10
20
30
50
100
HER2: -
+
-
+
-
+
promoter activity
Fold change in
C.
AG825: - + - + - +
MCF7-HER2
0.0
0.5
1.0
1.5
promoter activity
Fold change in
B.
NR6-HER2
AG825: - + - +
promoter activity
Fold change in
0.0
0.5
1.0
1.5
TBP-Luc
c-Fos-Luc
B3-Integrin-Luc
Figure 5. HER2 does not transcriptionally regulate TBP. A). Cells were transiently co-
transfected with 6 ug of a HER2 expression vector and 2 ug of the designated promoter reporter
construct. Cells were serum starved the following day and 6-8 hours later treated with AG825 (50
uM) overnight. Cells were harvested the following day and analyzed for luciferase activity. Fold
changes were calculated based on control (TBP promoter + empty expression vector), normalized
to total protein. B, C). Cells were transfected with the promoter reporter construct and serum
starved the following day. 6-8 hours post-serum starvation cells were treated with 50 uM AG825
overnight. Cells were harvested the following day and analyzed for luciferase activity and
normalized to total protein. The values shown are the mean ± SEM of at least 3 independent
determinations.
2.2 SUMMARY
Stable or transient expression of EGFR1 or EGFRvIII induces TBP
promoter activity leading to increased TBP protein levels. In contrast, stable or
transient expression of HER2 does not regulate TBP gene expression. Since the
intracellular region of EGFR1 and EGFRvIII is identical, it is not surprising that
both EGFR1 and EGFRvIII regulate the TBP promoter. However, the differences
in signal transduction mediated by HER2 responsible for its inability to regulate
TBP expression are not yet clear.
19
2.3 MATERIALS AND METHODS
2.3.1 Cell Culture
All NR6 cell lines were grown in Minimum Essential Media with
Earl’s salts (MEM) supplemented with Fetal Bovine Serum (50ml), Penicillin
(500 units), Streptomycin (500 ug), and Glutamine (500 units). All U87 cell lines
were grown in Dulbecco’s Modified Eagle Media-4.5g/L D-glucose (DMEM,
Mediatech, #10-013-CV) supplemented exactly as just described. NIH-3T3 cell
lines were grown in special DMEM media (Gibco #12430-054) and supplemented
as described above. MCF-7 cell lines were grown in RPMI-1640 media
supplemented as described above and additionally with 1.4 ml Insulin (Gibco
#13007-018 or Sigma I-0516). Media used for U87, NR6, and NIH-3T3 stable
cell lines were additionally supplemented with 600ug/ml G418. The NIH3T3-
EGFRvIII cell line was grown in media supplemented with 100ug/ml Kanamycin
in addition to the G418. Unless specifically noted, all media was obtained from
the USC microchemical cell culture core facility. All cell lines were grown at 37
C and 5% CO2. The NR6 parental cell line is null for EGFR1, HER2, EGFR3,
and EGFR4 expression (14). NR6 stable cell lines overexpressing EGFR1 or
EGFRvIII or HER2 were generated and characterized (1, 4). The NIH-3T3
parental cell line has very low endogenous expression of EGFR1 and is null for
HER2, EGFR3 and EGFR4 expression (14). NIH3T3 stable cell lines
overexpressing HER2 or EGFRvIII were generated and characterized (27, 40).
The U87 parental cell line has very low expression of endogenous EGFR1 and is
20
null for HER2, EGFR3, and EGFR4 (43). U87 stable cell lines overexpressing
EGFR1 or EGFRvIII were generated and characterized (41). The MCF-7 parental
cell line has low endogenous expression of EGFR1, medium expression of
EGFR3 and EGFR4, and no expression of HER2 (21, 23). MCF-7 stable cell line
overexpressing HER2 were generated and characterized (4).
2.3.2 Western Blot Analysis
Cells were seeded into a 10 cm dish so that they were 85-90% confluent
on the day of isolation. Chemical treatments were performed approximately 16
hours prior to protein isolation but 6-10 hours post-serum starvation where
designated, as described in the transient transfection protocol. Cells were washed
twice with ice-cold 1x PBS and 300-500 ul of lysis buffer (20mM Tris, pH 7.5,
150mM NaCl, 0.1% Triton X-100, 2.5 mM sodium pyrophosphate, 1mM β-
Glycerolphosphate, 1mM Na3VO4, 1mM PMSF, and 10ul/ml protein inhibitor
cocktail set III) were added. Cells were incubated on ice for 15 minutes and
transferred to a microcentrifuge followed by a 15 second sonnication. Lysates
were cleared by centrifugation at 10,000 g for 20 minutes at 4 °C. Supernatant
was aliquoted out and stored at -80 °C. Protein concentrations were determined
by the Bradford method using the Bio-Rad protein assay reagent (# 500-0006).
Briefly, 5 ul of cell lysate were added to 200 ul of 1X protein assay reagent and
read at 590 nm. Equal amounts of whole cell lysates were diluted with laemmli
sample buffer (1X final concentration) and samples were heated at 95 °C for 5
minutes. Samples were then subjected to SDS-polyacrylamide gel electrophoresis
21
(SDS-PAGE). After electrophoresis, proteins were transferred to Hybond-ECL
Nitrocellulose membrane (Amersham Biosciences, RPN 2020D) via wet transfer
and non-specific binding sites were blocked with either 5% non-fat milk in PBS
or 5% BSA in TBS for at least 1 hour. Immunoblotting was carried out following
the specific protocol provided by the antibody manufacturer. Antibodies
purchased from Cell Signaling: phospho-Tyr1173-EGFR1 (# 4407). BD
Biosciences: EGFR1 (# 610016). Antibodies purchased from Santa Cruz
Biotechnology: phospho-Tyrosine (sc-508). The HER2 antibody, 10H8, was a
kind gift from Dr. Michael Press (44). The human TBP antibody was purchased
from Upstate Biotechnology (# 06-241 ). The β-actin antibody was purchased
from Chemicon (# MAB501R ). Goat Anti-Mouse IgG Peroxidase conjugated or
Goat Anti-Rabbit IgG peroxidase conjugated secondary antibodies were
purchased from Pierce (# 31430 and # 31460, respectively). Chemiluminescence
reagent used for peroxidase detection of conjugated antibodies was purchased
from Pierce (# 1856136).
2.3.3 Plasmid DNAs
The plasmids used for transient expression of EGFR1 and EGFRvIII, p-
HβAPr-1neo-β-actin-EGFR1 (or EGFRvIII), were kindly provided by Dr. Darell
Bigner (1). All human wild type and ETS mutant TBP promoter-luciferase
constructs were a gift from Dr. Diane Hawley (16). The human β3-Integrin
luciferase reporter gene constructs was provided by Dr. Chi Dang (7). The human
CMV-β-galactosidase, and β-actin-Renilla reporter gene constructs were a gift
22
from Dr. Amy Lee. The plasmids used for transient expression of HER, p-SV2-
neo-LTR-HER2, or the empty vector construct, p-SV2-neo-LTR, were provided
by Dr. Michael Press (4). The human c-Fos-luciferase and ∆56c-Fos mutant
luciferase promoter constructs were provided by Dr. Craig Hauser (17).
2.3.4 Transient Transfections
The day before transfection 100,000 logarithmically growing cells were
seeded per 35-mm well of a 6-well plate containing 2 ml of growth medium.
Cells were kept at 37°C and 5% CO2. The following day, the transfection mixture
was prepared with 3 ug of DNA and 3 ul of F1 transfection reagent (Targeting
Systems, #001) in 250 ul of DMEM and incubated at 37 °C for 25 minutes. Each
mixture was then added to each well of the 6-well plate (250 ul transfection
mixture into 2 ml growth media). Approximately 24 hours post-transfection,
medium was removed by gentle aspiration and washed once with plain DMEM
followed by the addition of 2 ml of low serum media (0.5%). 6-10 hours post-
serum deprivation cells were treated with 2 uM AG1478 (Calbiochem #658552)
for mouse cell lines, or 10 uM AG1478 for human cell lines, or 25uM AG825
(Calbiochem #121765), or 50uM AG879 (Calbiochem #658460). EGF (Sigma #
E-4127) treatment (2-100ng/ml), where designated, started 6-10 hours post-serum
deprivation or 1-hour post-AG1478 treatment, where designated. After an
additional 12-15 hour incubation, cells were harvested and whole cell lysates were
prepared.
23
2.3.5 Preparation of Cell lysates and Luciferase Assay
The Promega protocol was followed for making lysates for luciferase and
beta-galactosidase (β-gal) assays. Briefly, 4 volumes of water were added to 1
volume of 5X reporter lysis buffer (Promega, # E397A). Media was aspirated
from each dish and cells were rinsed once with ice-cold phosphate buffered saline
(PBS). 50-100 ul of 1X reporter lysis buffer were added to each plate and placed
on ice for 10 minutes. Cells were scraped from each well and transferred to a 1.5
ml microcentrifuge tube and placed on ice. Cells were vortexed and incubated at
-80 °C for 10 min or alternatively left overnight. Cells were thawed quickly in a
37 °C water bath then vortexed and cleared by centrifugation at 10,000 x g for 20
minutes at 4 °C. Supernatant was collected and used for protein, luciferase
assays. For luciferase activity, 10-20 ul of cell lysates were added to 100 ul of
luciferase assay reagent (Promega, # E1501) in a luminometer tube. Mixture was
vortexed and tube was placed in the luminometer and read immediately. Protein
concentrations were analyzed by the Bradford method using the Bio-Rad protein
assay reagent (# 500-0006). Fold changes in promoter activities were calculated
based on control (reporter gene + empty expression vector) and normalized to
total protein. Standard deviations of the mean, SEM ±, were calculated using at
least 3 independent determinations. Fold changes in promoter activities were
calculated based on control (untreated or mismatch siRNA) and normalized to
total.
24
CHAPTER 3: Transcriptional regulation of TBP by EGFR1
A putative ETS response element within the TBP promoter located
approximately 50 bp upstream of the transcriptional start site was previously
shown to be important for TBP promoter induction by the MAPKs downstream of
oncogenic Ras and EGF stimulation (30, 64). The requirement of this site for
EGFR1-mediated regulation of TBP expression was extensively addressed here.
The Ets transcription factors regulate cell growth, apoptosis, differentiation,
developmental processes, and cellular transformation. They become activated by
kinases which enhances their DNA binding activity. The specific Ets factor
downstream of Ras that serves to regulate the TBP promoter was identified here.
The MAPKs consist of p38, ERK, and JNK. Each of these MAPKs consist of
several isoforms. p38 consists of α, β, γ, and δ isoforms. JNK consists of JNK1,
JNK2, and JNK3 isoforms. ERK consists of ERK1 and ERK2 isoforms and an
alternatively spliced form of ERK1, ERK1b. Emerging evidence by many
laboratories has recently shown that the different isoforms within each class of
MAPKs can have distinct biochemical functions. We specifically addressed the
involvement of JNK1, and JNK2 in regulating TBP expression because they are
ubiquitously expressed. The specific functions of JNK1 and JNK2, have been
controversial. In vitro data suggests that both JNK1 and JNK2 phosphorylate a
common Ets transcription factor, Elk-1 (61). However, these isoforms have also
been shown to have differences in their gene targets and substrate specificities (3,
22, 36, 47).
25
3.1 RESULTS
3.1.1 EGFR1 transcriptionally regulates TBP via a putative ETS site
TBP promoter mutant constructs containing a non-ETS consensus
recognition sequence were used to confirm the requirement of this site for EGFR1
mediated regulation of TBP expression (Figure 6A) (16). While transient
expression of EGFR1 in NR6 cells induced activation of the wild type TBP
promoter in an AG1478 sensitive manner, the ETS mutant construct had
significantly impaired response to EGFR1 activation and AG1478 treatment
(Figure 6B). The requirement of this putative ETS site was also confirmed in the
U87 parental cell line where transient expression of EGFR1 induced the wild type
TBP promoter construct by approximately 3-fold compared to a 1.5-fold induction
for the ETS mutant construct (Figure 6C).
The requirement of this ETS site was then addressed in the EGFR1 stable
cell lines using the same constructs as described above (Figure 6A). As expected,
the wild type TBP promoter was sensitive to AG1478 treatment but the ETS
mutant TBP promoter, however, displayed reduced promoter activity and was
insensitive to AG1478 treatment (Figure 6D). Similarly, a shorter fragment of the
TBP promoter (-230/+66) also showed a requirement for this ETS site for EGFR1
mediated regulation (Figure 6E). This data suggests that EGFR1 requires a
putative ETS response element to transcriptionally regulate TBP.
The minimal promoter necessary for EGFR1 mediated induction of TBP
promoter activity was determined using several TBP promoter deletion constructs.
26
B.
NR6
AG1478:
0
1
2
3
4
5
- - - - + +
EGFR1: + - - + + +
Fold change in
TBP promoter activity
C.
U87
EGFR1:
- - + +
0
1
2
3
4
Fold change in
TBP promoter activity
A.
hTBPEtsmut-Luc
hTBP-Luc
Luciferase
Luciferase
-736 -230 -84 +1 +66
-736 -230 -84 +1 +66
Ets
Ets
U87-EGFR1
0.00
- - + +
AG1478:
0.25
0.50
0.75
1.00
1.25
Fold change in -230/66
TBP promoter activity
D.
- - + + AG1478:
0.00
0.25
0.50
0.75
1.00
1.25
Fold change in -736/66
TBP promoter activity
U87-EGFR1
E.
EGF: + + + + + +
EGF: + + + +
+ + + + EGF:
+ + + +
EGF:
Figure 6. EGFR1 targets and ETS response element. A). Schematic of the -736/+66 genomic
fragment containing the human TBP promoter demonstrating the Ets site that is mutated in the
EtsmutTBP-Luc construct. B, C). Cells were transiently co-transfected with 4 ug of an EGFR1
expression vector and 2 ug of a promoter reporter construct. Cells were serum starved the
following day and 6-8 hours later treated with EGF (50 ng/ml) and/or 2 uM (NR6) or 10 uM
(U87) AG1478 overnight. Cells were harvested the following day and analyzed for luciferase
activity. Fold changes were calculated based on control (TBP promoter + empty expression
vector), normalized to total protein. D). Cells were transfected with a promoter reporter construct
and serum starved the following day. 6-8 hours post-serum starvation cells were treated with EGF
(50 ng/ml) and/or AG1478 (10 uM) overnight. Cells were harvested the following day and
analyzed for luciferase activity. E). Cells were transfected as described in “D” except a shorter
TBP promoter fragment (-230/+66 TBP-Luc) was used. The values shown are the mean ± SEM of
at least 3 independent determinations.
27
All TBP promoter luciferase reporter gene constructs used, as diagramed in Figure
7A, were transiently transfected into the U87-EGFR1 cells and treated with EGF
and/or AG1478.
The minimal promoter necessary for EGFR1 mediated induction of TBP
promoter activity was determined using several TBP promoter deletion constructs.
All TBP promoter luciferase reporter gene constructs used, as diagramed in Figure
7A, were transiently transfected into the U87-EGFR1 cells and treated with EGF
and/or AG1478. Each wild type construct corresponding to the promoter regions
-736/+66, -230/+66, -84/-1, or -54/-1 were induced by EGFR1 activation (Figure
7B). The ETS mutant constructs in context to the -736/+66, -230/+66, or -84/-1
TBP promoter regions were weakly induced by EGFR1 and showed insensitivity
to AG1478 treatment, further confirming the requirement of this response element
(Figure 7B). The minimal promoter for EGFR1-mediated induction lies within
the -54/-1 promoter fragment and requires an intact ETS site.
28
0.0
Fold change in
TBP promoter activity
0.3
0.6
0.9
1.2
+
AG1478:
-84/-1 hTBP-Luc
-84 -1
Ets Luciferase Ets
-84/-1 hTBPEtsmut-Luc
-84 -1
Ets
Luciferase Ets
-54/-1 hTBP-Luc
-54 -1
Ets Luciferase
Ets
-736/+66 hTBP-Luc
Luciferase
-736 -230 -84 -1 +66
Ets Ets
-736/+66 hTBPEtsmut-Luc
-736 -230 -84 -1 +66
Luciferase Ets Ets
-230/+66 hTBP-Luc
Luciferase
-230 -84 -1 +66
Ets
Ets
-230/+66 hTBPEtsmut-Luc Luciferase
-230 -84 -1 +66
Ets
Ets
A. B.
-
+
-
+
-
+
-
+
-
+
-
+
-
+
EGF:
+
+
+
+
+
+
+
+
+
+
+
+
+
Figure 7. EGFR1 requires sequences within -54/-1 TBP promoter fragment. A). Diagram of
TBP promoter deletion constructs used to determine the minimal promoter required for EGFR1-
mediated regulation. B). U87-EGFR1 cells were transiently transfected with a promoter reporter
construct. Cells were serum starved the following day and 6-8 hours later treated with EGF (50
ng/ml) and/or AG1478 (10 uM) overnight. Cells were harvested the following day and analyzed
for luciferase activity. Fold changes were calculated based on untreated control, normalized to
total protein.
3.1.2 Transcriptional regulation of TBP by EGFR1 requires Ras-MAPK
EGF stimulation of JB6 cells induces TBP promoter activity in a Ras
dependent manner and requires the activation of p38, ERK, and JNK (30, 64)
(Figure 8A). Consistent with these results, treatment of U87-EGFR1 cells with
inhibitors for p38, ERK, and JNK each reduced TBP promoter activity (Figure
8B). Each of the three classes of MAPKs consists of several isoforms. To begin
to determine which of the specific MAPK isoforms regulate TBP gene expression,
we first defined the roles of the different JNK isoforms.
29
EGFR1
Ras
p38 JNK ERK
TBP gene expression
EGF
U87-EGFR1
Fold change in
TBP promoter activity
0.00
0.25
0.50
0.75
1.00
1.25
+
ERK inhibitor:
JNK inhibitor:
p38 inhibitor:
-
-
-
+
+
-
-
-
-
-
-
A. B.
EGF:
+ + + +
Figure 8. EGFR1 requires MAPKs to transcriptionally regulate TBP. A). Schematic of the
signal transduction pathway downstream of EGF stimulation previously reported in JB6 cells. B).
U87-EGFR1 cells were transiently transfected with 2 ug of a promoter reporter construct. Cells
were serum starved the following day and 6-8 hours later treated with EGF (50 ng/ml) and/or
MAPK inhibitor (U0126, 30uM; JNK inhibitor V , 80 uM; SP220025(p38), 50 uM) overnight.
Cells were harvested the following day and analyzed for luciferase activity. Fold changes were
calculated based on untreated control, normalized to total protein. The values shown are the mean
± SEM of at least 3 independent determinations.
The specific functions of the ubiquitously expressed JNKs, JNK1 and JNK2, have
been controversial. In vitro data suggests that both JNK1 and JNK2
phosphorylate a common Ets transcription factor, Elk-1 (61). However, these
isoforms have also been shown to have differences in their gene targets and
substrate specificities (3, 22, 36, 47). We addressed the specific role of JNK1 and
JNK2 in regulating TBP promoter activity. This study was initially addressed in a
human hepatoma cell line, Huh7, which expresses moderate levels of EGFR1 and
30
no HER2 (Figure 9D). We tested the effect of transiently modulating the levels of
JNK expression on TBP expression with RNA interference. Transient expression
of siRNAs specific for JNK1 or JNK2 selectively decreased JNK1 or JNK2
protein levels compared to control cells transfected with control siRNA (mm)
(Figure 9A). Decreased JNK1 expression resulted in a decrease in TBP
expression, whereas, decreased JNK2 expression resulted in an increase in TBP
expression (Figure 9A). Collectively, this data along with our published work
(63) supports a non-cell type specific differential regulation of the JNKs on TBP
expression. To define the mechanism for JNK1 and JNK2-mediated EGFR1
regulation of TBP promoter activity, we first addressed the effects of modulating
JNK expression on TBP promoter activity. The wild type TBP promoter retained
EGF responsiveness in the presence of mm siRNAs but promoter induction was
abolished when JNK1 expression was decreased (Figure 9B). Reducing the
expression of JNK1, however, had no effect on the wild type promoter in
unstimulated cells. We next tested whether JNK1 functioned through the ETS site
required for EGFR1 induction of TBP promoter activity as described above.
Mutation of the Ets site prevented EGF mediated stimulation of the promoter, and
promoter activity was insensitive to decreased JNK1 protein levels (Figure 9B).
Conversely, decreased JNK2 expression resulted in an induction of the wild type
TBP promoter in both unstimulated and EGF stimulated cells (Figure 9C).
Activation of the ETS mutant promoter was unaffected by decreased JNK2
expression (Figure 9C).
31
C.
D.
EGFR1
α-EGFR1
α-HER2
0
1
2
3
4
5
JNK1 siRNA:
EGF:
Fold change in
TBP promoter activity
- + + + + -
- -
-
+ +
-
- -
+
+ 0
1
2
3
4
5
JNK2 siRNA:
EGF:
Fold change in
TBP promoter activity
- + + + + -
- -
-
+ +
-
- -
+
+ TBP-Luc
TBPEtsmut-Luc
A.
B.
siRNA: mm JNK1 JNK2 mm JNK1JNK2
0.0
Fold change in
JNK protein levels
0.5
1.0
1.5
α-JNK1
α-JNK2
siRNA: mm JNK1 JNK2
JNK1
JNK2
TBP
β
-actin
Fold change in
TBP protein levels
0
1
2
3
α-TBP
mm JNK1 JNK2
Huh7
Figure 9. TBP expression is differentially regulated by the JNKs. A). Huh7 cells were
transfected with an siRNA selective for JNK1, JNK2, or mismatch siRNAs. Protein lysates
derived from the siRNA-transfected cells were subjected to immunoblot analysis using antibodies
against specific JNKs, β-actin, or TBP (left panel). Fold changes in protein levels were calculated
by normalizing the representative quantified TBP, JNK-1, or JNK-2 protein bands to the
representative quantified β-actin protein bands (right panel). B, C). Huh7 cells were transfected
with the promoter construct designated and cells were co-transfected with mismatch siRNA (-) or
JNK1 siRNA (+) “B” or mismatch siRNA (-) or JNK2 siRNA (+) “C”. Cells were serum starved
24 hours post-transfection and where designated, treated with EGF (50 ng/ml) 6-8 hours later for
overnight. Protein lysates were prepared and luciferase activity was measured. Fold changes were
calculated based on control (TBP promoter + mm siRNA) and normalized to total protein. D).
Immunoblot analysis of Huh7 derived WCL using antibodies against EGFR1 or HER2. The
values shown are the mean ± SEM of at least 3 independent determinations.
32
Collectively, this data suggests that both JNK1 and JNK2 target the putative ETS
site to differentially regulate TBP gene expression.
3.1.3 Elk-1 is targeted to the TBP promoter by EGFR1
The requirement for a putative ETS site for EGFR1-mediated TBP
promoter induction begs the question as to which specific Ets factor is bound.
The recruitment of Elk-1 to DNA requires Elk-1 to be phosphorylated. In
addition, JNK1, JNK2, p38, and ERK have all been shown to phosphorylate
Elk-1, in vitro, (13, 32, 61). Given that EGFR1 requires the activation of p38,
ERK, and JNK1 to regulate TBP expression and Elk-1 is ubiquitously expressed,
we determined whether Elk-1 was bound to the TBP promoter. Inhibition of
Elk-1 expression by RNA interference was used to determine the requirement of
this transcription factor for EGFR1-mediated TBP promoter induction. Transient
expression of Elk-1 siRNAs downregulated Elk-1 protein levels which
corresponded to a significant decrease in TBP protein levels (Figure 10A)
compared to cells transfected with mm siRNA. To test the requirement of Elk-1
for EGFR1-mediated transcriptional regulation of TBP, Huh7 cells were
transiently transfected with a wild type or ETS mutant TBP promoter construct
together with Elk-1 siRNAs or mm siRNAs. EGF stimulation of the TBP
promoter was abolished when Elk-1 protein levels were decreased (Figure 10B).
Decreased Elk-1 expression in the absence of EGF stimulation did not affect TBP
promoter activity (Figure 10B) consistent with the requirement of Elk-1 to be
phosphorylated in order to stably bind to DNA. The ETS mutant construct was
33
used to identify whether Elk-1 was targeted to this particular putative ETS site.
EGF stimulation and decreased Elk-1 expression had no effect on the ETS mutant
promoter activity suggesting that EGFR1 targets Elk-1 to this specific putative
ETS site (Figure 10B). The requirement for Elk-1 was also confirmed in the U87-
EGFR1 cell line by modulating Elk-1 levels by siRNAs (Figure 10C). Decreased
Elk-1 protein levels prevented EGFR1 mediated activation of the TBP promoter
in the U87 cells, consistent with the Huh7 observations (Figure 10D).
Chromatin immunoprecipitation (ChIP) assays were performed to
determine if Elk-1 was directly involved in EGFR1 mediated induction of TBP
promoter activity. Primers were designed to target the promoter region containing
the required ETS site and, as a negative control, primers were designed that
targeted an upstream region of the promoter (Figure 11A). Serum starved Huh7
cells were treated with or without EGF for 5 minutes prior to crosslinking and
isolation. Primers targeting the TBP promoter sequences containing the ETS site
of interest showed that activation of EGFR1 increased Elk-1 recruitment to the
TBP promoter (Figure 11B). Primers amplifying an upstream region of the
promoter similarly amplified input chromatin but failed to amplify the Elk-1
immunoprecipitated chromatin, confirming the specificity of the results obtained
with the target primers (Figure 11D). Real time qPCR of the Elk-1
immunoprecipitated chromatin showed a similar increase in Elk-1 recruitment
with EGF treatment as conventional PCR using the same samples (Figure 11C).
34
A.
C.
B.
0
1
2
3
4
5
Fold change in
TBP promoter activity
Elk-1 siRNA:
EGF:
-
+ + + + -
- -
-
+ +
-
- -
+
+ Huh7
TBP-Luc
TBPEtsmut-Luc
D.
Elk-1
TBP
β-actin
siRNA: mm Elk-1
TBP promoter activity
0.4
0.8
1.2
Fold change in
Elk-1 siRNA: + -
0.0
U87-EGFR1
EGF: + +
Elk-1
β-actin
siRNA: mm Elk-1
Figure 10. Elk-1 regulates TBP expression. A). Huh7 cells were transfected with either
mismatch siRNA or Elk-1 siRNA. Immunoblot analysis was performed using lysates prepared
from transfected cells using antibodies against Elk-1, TBP, and -actin. B). Huh7 cells were co-
transfected with the designated siRNAs and either the wild type promoter or Ets mutant TBP
promoter construct. Cells were serum starved 24 ho urs post-transfection and treated with EGF
(50 ng/ml) 6-8 hours later for overnight. Fold changes were calculated based on control (TBP
promoter + mm siRNA) and normalized to total protein. C). U87-EGFR1 cells were transfected
with either mismatch siRNA or Elk-1 siRNA. Immunoblot analysis was performed using lysates
prepared from transfected cells using antibodies against Elk-1, and -actin. D). U87-EGFR1 cells
were treated and analyzed as described in “B”. The values shown are the mean ± SEM of at least 3
independent determinations.
35
-66 +5 -119 -3158 -3315 -4500
Ets
ChIP Antibody:
0
10
20
Elk-1 Occupancy
EGF: - +
Elk-1
+
IgG
A.
B.
C.
3313/-3158 primers
EGF:
Template:
ChIP Antibody:
-
+ +
- - + + - -
+
IgG Elk-1 Input NTC
Template:
-119/+5 primers
IgG Elk-1 Input NTC
EGF:
- + + -
- + + - - + + -
D.
Figure 11. Elk-1 directly modulates EGF-induced TBP promoter activity and is differentially
regulated by JNK1 and JNK2. A). Schematic diagram of a genomic fragment containing the
human TBP promoter. The arrows depict the relative location of the PCR primers used for the
ChIP analysis. B). Chromatin immunoprecipitation (ChIP) assays were performed as described in
“Materials and Methods”. Resultant chromatin was immunoprecipitated with Elk-1 antibody or a
control antibody. Specific DNA fragments were quantified by semi-quantitative PCR using
primers targeting the regions within the TBP promoter specified in “A”. “Input” represents
primer-specific amplification of 10% of total chromatin isolated for each sample. “NTC”
designates reactions performed without added template. C). qPCR of DNA in “B” using the
same primers. D). ChIP analysis as described in “B” except that the upstream negative control
primers were used as shown in “A”.
36
0
1
2
3
4
5
6
7
Occupancy on TBP promoter
-119/+5 primers -3350/-3315 primers
EGF:
siRNA:
ChIP Antibody:
Elk-1 IgG
mm JNK1 JNK2
+ - + - + - + - +
IgG Elk-1
mm mm
Huh7 D.
siRNA: mm JNK2 JNK1
Elk-1
β-actin
EGF:
- + -
+
mm
E.
Figure 11. cont. Elk-1 directly modulates EGF-induced TBP promoter activity and is
differentially regulated by JNK1 and JNK2. (D). Huh7 cells were transfected with mismatch,
JNK1, or JNK2 siRNAs. Cells were serum starved and, where designated, treated with EGF (50
ng/ml). ChIP assays were performed using the transfected cells and qPCR was performed to
quantify the amplified DNA. The fold change in TBP occupancy was calculated based on control
(TBP promoter + mm siRNA). The results shown were derived from three independent chromatin
preparations. (E). Immunoblot analysis was performed using lysates derived from Huh7 cells
transfected with mm, JNK2 or JNK1 siRNA (left panel).
How JNK1 and JNK2 directly affect Elk-1 TBP promoter occupancy was
tested by ChIP analysis on cells transiently transfected with mismatch, JNK1, or
JNK2 siRNA in the presence or absence of EGF stimulation. A loss of JNK1
expression resulted in a loss of Elk-1 recruitment to the TBP promoter in EGF
stimulated cells (Figure 11D). A loss of JNK2 expression, however, enhanced
Elk-1 recruitment to the promoter independent of EGFR1 activation (Figure 11D).
Again, the control primer set confirms the validity of these results (Figure 11D).
This data suggests that activated JNK1 recruits Elk-1 to the TBP promoter,
37
A.
B.
D.
C.
+ - - +
0.0
Fold change in TBP
promoter activity
0.5
1.0
1.5
c-Fos siRNA:
- +
mm siRNA:
- -
c-Jun siRNA:
+ - - +
+ AG1478:
0.0
0.5
1.0
1.5
Fold change in TBP
promoter activity
+ - -
Elk-1
AP-1
actggcggaagtgacattatcaacgcgcgccagggttcagtgaggtcgggca
-54
-1
hTBP-Luc Luciferase
-230 -84 -1 +66
Ets AP1
- hTBPAP1mut-Luc Luciferase
-230 -84 -1 +66
Ets AP1
-736
-736
-3358/-3315 primers
TBP promoter occupancy
AG1478:
-119/+5 primers
TBP promoter occupancy
Elk-1 c-Jun c-Fos IgG
+ + + +
+ + + -
+ + +
- - -
0
2
4
6
8
10
12
14
Elk-1 c-Jun c-Fos
+ + + - + - + - +
+ + + - - - - - -
0
2
4
6
8
10
12
14
EGF:
ChIP Antibody:
c-Fos
β-actin
c-Fos siRNA:
+
-
c-Jun
β-actin
c-Jun siRNA:
+
-
+ EGF: + +
+
+ EGF: + +
+
Figure 12. EGFR1 does not target and adjacent AP-1 site. A). Schematic of the annotated TBP promoter containing
an adjacent AP-1 site (top). Schematic of the wild type and AP-1 mutant TBP promoter reporter constructs used for
promoter assays (bottom). B). U87-EGFR1 cells were transiently transfected with 2 ug of the wild type promoter or
AP-1 mutant promoter reporter construct. Cells were serum starved the following day and 6-8 hours later treated with
EGF (50 ng/ml) and/or AG1478 (10 uM) overnight. Cells were harvested the following day and analyzed for luciferase
activity. Fold changes were calculated based on untreated control, normalized to total protein. C). U87-EGFR1 cells
were transiently co-transfected with 2 ug of the wild type TBP promoter reporter construct and either mismatch siRNA
(-), c-Jun siRNA (+), and/or c-Fos siRNA (+). Fold changes were calculated based on control (TBP promoter + mm
siRNA) and normalized to total protein (left panel). Immunoblot analysis was performed using lysates prepared from
siRNA transfected cells using antibodies against c-Fos, c-Jun, and -actin (right panel). D). ChIP assays were
performed using the previously described primer sets on serum starved U87-EGFR1 cells treated with EGF (50 ng/ml)
and AG1478 (10 uM) overnight where designated. qPCR was performed to quantify the amplified DNA. The fold
change in TBP occupancy was calculated based on untreated control. The values shown are the mean ± SEM of at least
3 independent determinations.
38
whereas JNK2 prevents Elk-1 recruitment to the TBP promoter therefore inhibiting TBP
promoter induction, independent of stimuli. Modulating JNK1 or JNK2 protein levels
had no effect on Elk-1 expression levels (Figure 11E), suggesting that JNK1 and JNK2
are regulating the activity or recruitment of Elk-1 to the promoter.
To test whether other response elements within the TBP promoter are
targeted by EGF, the minimal TBP promoter sequence was annotated for potential
transcription factor binding sites. Promoter annotation revealed an adjacent AP-1
site downstream of the Elk-1 site (Figure 12A). The requirement of this AP-1
element was determined by site-directed mutational analysis to generate point
mutations within the AP-1 site to abolish the AP-1 consensus recognition
sequence. EGFR1-mediated induction of the TBP promoter was unaffected by the
loss of the AP-1 binding site, as both the wild type promoter and the AP-1 mutant
promoter were activated in U87-EGFR1 cells (Figure 12B). To further confirm
the lack of requirement of the AP-1 regulatory promoter element, AP-1 specific
siRNAs were transiently transfected into U87-EGFR1 cells. A decrease in the
protein levels of two AP-1 members known to be activated by EGFR1, c-Jun and
c-Fos, did not appreciably affect TBP promoter activity (Figure 12C, Left). This
was independent of individual or co-expression of c-Jun and c-Fos siRNAs. The
efficacy of the c-Jun and c-Fos siRNAs was confirmed by immunoblot analysis
(Figure 12C, Right).
To confirm that the recruitment of Elk-1, but not c-Jun or c-Fos, to the
TBP promoter by EGFR1 is not cell type specific, ChIP analysis was repeated in
39
U87-EGFR1 cells. Consistent with the Huh7 data, Elk-1 recruitment was
increased with EGF treatment and decreased with AG1478 treatment (Figure 12D,
Left). There was no EGFR1-specific targeting of c-Jun or c-Fos recruitment
observed (Figure 12D, Left). The control primer set confirmed the validity of
these results(Figure 12D, Right).
3.2 SUMMARY
EGFR1 induction of TBP promoter activity requires the activation of Ras,
p38, JNK1 and ERK to recruit Elk-1 to the TBP promoter. Many promoters are
regulated by ETS/AP-1 composite sites but EGFR1 under our conditions does not
make use of an adjacent AP-1 response element. We next addressed the
importance of the ETS and AP-1 sites for EGFRvIII-mediated regulation of TBP
gene expression.
3.3 MATERIALS AND METHODS
3.3.1 Cell Culture
All NR6 and U87 cell lines were described in Chapter 2. Huh7
cells were grown in Dulbecco’s Modified Eagle Media-4.5g/L D-glucose
(DMEM, Mediatech, #10-013-CV) supplemented exactly as just described.
NIH-3T3 cell lines were grown in special DMEM media (Gibco #12430-054) and
supplemented as described above. All cell lines were grown at 37 C and 5% CO2.
40
3.3.2 Western Blot Analysis
Cells were seeded into a 10 cm dish so that they were 85-90% confluent
on the day of isolation. Chemical treatments and isolation of lysates was
described in Chapter 2. Samples were prepared for SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) and immunoblot analysis as described in Chapter 2.
Antibodies purchased from Cell Signaling: c-Fos (# 4384), c-Jun (# 9162, #
9165), Elk-1 (# 9182). BD Biosciences: JNK1/JNK2 (# 55425). Antibodies
purchased from Santa Cruz Biotechnology: Elk-1 (sc-355). JNK1 (# sc-1648),
Brf (# sc-17465), IgG (# sc-2027). The human TBP antibody was purchased from
Upstate Biotechnology (# 06-241 ). The β-actin antibody was purchased from
Chemicon (# MAB501R ). Goat Anti-Mouse IgG Peroxidase conjugated or Goat
Anti-Rabbit IgG peroxidase conjugated secondary antibodies and
chemiluminescence reagent were described in Chapter 2.
3.3.3 Plasmid DNAs
All human wild type and ETS mutant TBP promoter-luciferase constructs
were a gift from Dr. Diane Hawley (16) except for mutations made in the AP-1
response element, which is described under site-directed mutagenesis. The
human CMV-β-galactosidase reporter gene construct was a gift from Dr. Amy
Lee. The human c-Fos-luciferase promoter construct was provided by Dr. Craig
Hauser (17).
41
3.3.4 Transient Transfections
Cell plating, transfection, AG1478 treatment conditions, and lysate
isolation were described in Chapter 2. Where designated cells where transfected
with .1 ug of CMV- βgal reporter plasmid. Where designated cells were treated
with p38 inhibitor (Calbiochem # SB220025), JNK inhibitor V (Calbiochem #
420129), MEK inhibitor ( Promega # U0126) overnight and then isolated as
previously described.
For transient transfections of synthesized human small interfering RNA
(siRNA) oligonucleotide sequences, 150,000 cells/ 35mm dish were plated the
night before transfection and when in combination with promoter assays, siRNA
transfection occurred 4 hours post-plasmid DNA transfection. The Targeting
Systems siRNA kit (# 0060) was used. To 1 ml of DMEM per 35mm dish, 100nM
siRNA, 5 ul of solution A, and 15 ul of solution B was added and incubated in a
37 °C water bath for 25 minutes. During the 25 minute incubation, the cells were
washed twice with DMEM and the entire 1 ml of transfection mixture was added
to each dish. Cells were incubated at 37 °C and 5% CO2 for 2 hours and then 2
ml of complete growth DMEM medium was added and cells were incubated at 37
°C and 5% CO2 overnight. The next day the transfection medium was removed
and fresh growth medium was added. The next day the medium was removed and
cells were washed once with DMEM and 2 ml of low serum media was added
(0.5%). Cells were harvested the following day for luciferase activity or
immunoblot analysis. The siRNA oligonucleotide sequences were described
42
previously for JNK1 (11) , JNK2 (10), and Elk-1 (60). ON-TARGETplus duplex
c-Jun (# J-003268-10 and J-003268-12) and c-Fos (# J-003265-09 and
J-003265-10) siRNAs were purchased from Dharmacon.
3.3.5 Preparation of Cell lysates for Protein, Luciferase and β-gal Assays
The protein and luciferase assay protocol was described in Chapter 2.
Where designated, β-gal assays were performed in a 96-well plate to measure
transfection efficiency. β-gal activity was determined by incubating 50 ul of
diluted cell lysate with 50 ul of 2X assay buffer (200mM sodium phosphate
buffer, pH7.3, 3.2 mM MgCl2, 200mM β-mercaptoethanol and 1.33 mg/ml
ONPG) at 37 ºC for 30 minutes or until a faint yellow color develops. Reaction
was stopped by adding 150 ul of 1M Sodium Carbonate. Absorbance was read at
420 nm. To normalize for transfection efficiency luciferase readings were divided
by their respective β-gal reading.
3.3.6 Site-directed mutagenesis
Site-directed mutagenesis of the -736/+66, -230/+66, and -4500/+66 TBP
promoter-luciferase constructs were performed following Stratagene’s protocol
for QuickChange Site-Directed Mutagenesis Kit (cat # 200518, or 200521-
QuickChange II XL Site-Directed Mutagenensis Kit). Primers used for the ETS
(G-47A) mutation are as follows, FWD: GAA CTG GCA GAA GTG ACA TTA
TCA ACG CGC GCC AG, REV: CTG GCG CGC GTT GAT AAT GTC ACT
TCT GCC AGT TC. To change the wild type AP-1 recognition sequence
43
(GTGACATTAT) to a non-AP-1 recognition sequence at positions -39-38
(GTGATGTTAT) the following primers were used, FWD: GAA CTG GCG
GAA GTG ACT CTA TCA ACG CGC GCC AG; REV: CTG GCG CGC GTT
GAT AGA GTC ACT TCT GCC AGT TC. To create the double ETS and AP-1
mutant the following primers were used, FWD: GAA CTG GCA GAA GTG ACT
CTA TCA ACG CGC GCC AG; REV: CTG GCG CGC GTT GAT AGA GTC
ACT TCT GCC AGT TC. Sequencing confirmed that the appropriate mutation
was generated using Promega GL primer 1 (TGT ATC TTA TGG TAC TGT AAC
TG) and GL primer 2 (CTT TAT GTT TTT GGC CTC TTC CA), which are
specific for the pGL2 basic construct targeting the upstream and downstream
promoter insertion sites, respectively.
3.3.7 Real time PCR
Real-time PCR was performed using SYBR Green Supermix (Biorad #
170-8882) on a MX3000P system (Stratagene). The reaction mixture contained
0.15 nM of each primer, 1X SYBR master mix, 2ul template, and water for a final
reaction volume of 25 ul. TBP primer sequences were: -119 forward primer: 5’-
GAC CTA TGC TCA CAC TTC TCA TGG-3’; +5 Reverse primer: 5’-GAA CCT
GCC CGA CCT CAC TGA A-3’; -3315 Forward primer: 5’-CAG GAG TTG
GAG GTT GCA GT-3’; -3158 Reverse primer: 5’-GGC AAC TCA AGA CAG
CTA GCA A-3’. All Ct values were normalized to the PCR efficiency using the 1/
(2*PCR efficiency)
Ct
calculation. Normalized Ct values for antibody pull-downs
44
were normalized to input using Antibody IP*10/Input calculation and IgG. Fold
changes in promoter occupancy were calculated by setting the level of promoter
occupancy in the cells transfected with mismatch siRNA and in the absence of
EGF treatment at 1.
3.3.8 Chromatin Immunoprecipitation Assay
Chromatin immunoprecipitation (ChIP) assays were performed as described
in (62). Briefly, cells were treated with 100 ng/ml EGF for 5 min prior to cross-
linking with 1% formaldehyde. The cells were then lysed and chromatin was
sheared into 200-500 base pair fragments by sonnication. Chromatin was
immunoprecipitated with antibody rotating overnight. The immunoprecipitated
chromatin fragments were purified by immunoadsorption and elution from protein
A/G Plus Agarose beads (3 hours incubation following overnight IP). The cross-
links were reversed by incubating the DNA with 0.3M NaCl at 65°C overnight.
Proteins were digested with 0.1 ug/ul Proteinase K treatment for 2-3 hours at 45
ºC. Eluents were desalted and concentrated following the Qiaex II (Qiagen)
protocol (# 20021). The DNA region cross-linked to the protein was determined
by real time-qPCR.
45
CHAPTER 4: Transcriptional regulation of TBP by EGFRvIII
EGFR1 and EGFRvIII share the same C-terminal tails and activation of
signal transduction pathways is dictated, in part, by the intracellular domains.
Therefore, EGFR1 and EGFRvIII have the potential to activate similar upstream
intracellular signaling molecules. Results thus far indicate that both EGFR1 and
EGFRvIII transcriptionally regulate TBP expression. Since EGFRvIII and
EGFR1 both activate Ras and the MAPKs (40, 45), it was determined whether the
Elk-1 response element was similarly required for EGFRvIII-mediated TBP gene
regulation. EGFRvIII mediated signal transduction pathways have been shown,
however, to to have altered EGFR1 mediated signaling. Given this we
determined the minimal TBP promoter fragment and the involvement of the
putative response elements within this fragment.
4.1 RESULTS
4.1.1 EGFRvIII does not require the Elk-1 response element
An EGFRvIII expression vector together with either the wild type or ETS
mutant TBP promoter construct (Figure 13A) were transiently co-transfected into
the U87 parental cells to determine the requirement of this ETS site. Transient
expression of EGFRvIII induced activation of both the wild type and ETS mutant
promoter constructs compared to the vector control in U87 cells (Figure 13B). To
rule out cell type specific effects, NR6 cells were also co-transfected with these
vectors. In NR6 cells, EGFRvIII similarly activated both the wild type and ETS
46
mutant promoters in an AG1478 sensitive manner (Figure 13C). The
dispensability of this Elk-1 response element was confirmed in the U87-EGFRvIII
stable cell lines; both the wild type and ETS mutant promoters were activated by
EGFRvIII in these cells (Figure 13D). The Elk-1 independent regulation of TBP
expression by EGFRvIII was also addressed in two additional EGFRvIII stable
cell lines, NR6-EGFRvIII and NIH3T3-EGFRvIII. Inhibition of EGFRvIII
signaling significantly reduced activity of the ETS mutant promoter in both the
NR6-EGFRvIII cells (Figure 13E) and the NIH3T3-EGFRvIII cells (Figure 13F).
This data confirms that EGFRvIII-mediated induction of the TBP promoter is
Elk-1 independent.
4.1.2 EGFRvIII retains responsiveness with a -54/-1 TBP minimal promoter
To begin to identify the differences in signal transduction between
EGFRvIII and EGFR1, the requirement of ERK, JNK, and p38 were addressed
using chemical inhibitors. Inhibiting each of the three MAPKs abolished
EGFRvIII mediated effects on the TBP promoter. Thus, differences in the
requirement for MAPK induction by EGFR1 and EGFRvIII did not explain
differences in the requirement for the Elk-1 binding site (Figure 14A). The TBP
promoter response elements required for EGFRvIII mediated TBP promoter
regulation were determined by using several deletion constructs as previously
described and diagramed in Figure 14B. The -736/+66 and-230/+66 wild type
and ETS mutant constructs all showed similar induction levels and inhibition by
47
AG1478 treatment (Figure 14C). The short -84/-1 and -54/-1 promoter fragments
also retained EGFRvIII mediated induction (Figure 14C).
48
A.
hTBPEtsmut-Luc
hTBP-Luc
Luciferase
Luciferase
-736 -230 -84 +1 +66
-736 -230 -84 +1 +66
Ets
Ets
D. E.
U87-EGFRvIII
Fold change in
TBP promoter activity
AG1478: - + - +
0.0
0.5
1.0
1.5
AG1478: - + - +
0.0
0.5
1.0
1.5
NR6-EGFRvIII
Fold change in
TBP promoter activity
AG1478:
NIH3T3 NIH3T3-EGFRvIII
- + - +
0.0
0.5
1.0
1.5
2.0
Fold change in
TBP promoter activity
F.
B. C.
EGFRvIII:
Fold change in
TBP promoter activity
U87
- + - +
0
4
1
2
3
NR6
Fold change in
TBP promoter activity
EGFRvIII:
AG1478:
0
1
2
3
4
- + - + - + - +
- - + + - - + +
Figure 13. EGFRvIII transcriptionally regulates TBP via a non-ETS response element. A). Schematic diagram of the
-736/+66 genomic fragment containing the human TBP promoter. B, C). Cells were transiently co-transfected with 4
ug of an EGFRvIII expression vector and 2 ug of a promoter reporter construct. Cells were serum starved the
following day and 6-8 hours later treated with 2 uM (NR6) or 10 uM (U87) AG1478 overnight. Cells were harvested
the following day and analyzed for luciferase activity. Fold changes were calculated based on control (TBP promoter +
empty expression vector), normalized to total protein. D-F). Cells were transfected with a promoter reporter construct
and serum starved the following day. 6-8 hours post-serum starvation cells were treated with AG1478 (10 uM)
overnight. Cells were harvested the following day and analyzed for luciferase activity and normalized to total protein.
The data represents at least 3 independent experiments.
49
This supports the idea that EGFRvIII targets response elements contained within
the -54/-1 TBP promoter region.
The -54/-1 TBP promoter fragment contains both the Elk-1 binding site
and an overlapping AP-1 consensus sequence. Elk-1 protein levels were
decreased in U87-EGFRvIII cells to address the possibility that EGFRvIII may
target another putative ETS site within the TBP promoter. Immunoblot analysis
confirmed that Elk-1 protein levels were reduced in cells transiently transfected
with Elk-1 siRNA (Figure 14D, Right), however, decreased Elk-1 protein levels
had no effect on TBP promoter activity (Figure 14D, Left). As a positive control,
the human c-Fos promoter activity was analyzed under reduced Elk-1 expression
conditions (Figure 14D, Left). This data further supports the idea that EGFRvIII
targets the TBP promoter in an Elk-1 independent manner.
50
B. C.
U87-EGFRvIII
0.00
0.25
0.50
0.75
1.00
1.25
Fold change in TBP
promoter activity
+
ERK inhibitor:
JNK inhibitor:
p38 inhibitor:
-
-
-
+
+
-
-
-
-
-
-
A.
D.
TBP-Luc
c-Fos-Luc
Elk-1
β-actin
Elk-1 siRNA: -
promoter activity
Elk-1 siRNA: - -
Fold change in
0.00
0.25
0.50
0.75
1.00
1.25
U87-EGFRvIII
+
+
+
-54/-1 hTBP-Luc
-54 -1
Ets Luciferase Ets
-736/+66 hTBP-Luc Luciferase
-736 -230 -84 -1 +66
Ets Ets
-736/+66 hTBPEtsmut-Luc Luciferase
-736 -230 -84 -1 +66
Ets Ets
-230/+66 hTBP-Luc Luciferase
-230 -84 -1 +66
Ets Ets
-84/-1 hTBP-Luc
-84 -1
Ets Luciferase Ets
Fold change in
AG1478:
+
-
+
-
+
-
+
-
+
-
+
-
0.0
0.5
1.0
1.5
2.0
TBP promoter activity
-230/+66 hTBPEtsmut-Luc Luciferase
-230 -84 -1 +66
Ets Ets
Figure 14. EGFRvIII requires sequences within -54/-1 TBP promoter fragment to transcriptionally
regulate TBP. A). U87-EGFRvIII cells were transiently transfected with 2 ug of the designated promoter
reporter construct. Cells were serum starved the following day and 6-8 hours later treated with a MAPK
inhibitor (U0126, 30uM; JNK Inhibitor V , 80 uM; SB220025 (p38), 50uM) overnight. Cells were harvested
the following day and analyzed for luciferase activity. Fold changes were calculated based on untreated
control, normalized to total protein. B). Diagram of TBP promoter deletion constructs used to determine
the minimal promoter required for EGFR1-mediated regulation. C). U87-EGFRvIII cells were transiently
transfected with 2 ug of a promoter reporter construct. Cells were serum starved the following day and 6-8
hours later treated with AG1478 (10 uM) overnight. Cells were harvested the following day and analyzed
for luciferase activity. Fold changes were calculated based on untreated control, normalized to total protein.
The values shown are the mean ± SEM of at least 3 independent determinations. D). EGFRvIII cells were
co-transfected with mismatch or Elk-1 siRNA and either the TBP promoter or c-Fos promoter construct.
Cells were serum starved 24 hours post-transfection and the following day the resultant lysates were
analyzed for luciferase activity. Fold changes were calculated based on control (TBP promoter + mm
siRNA) and normalized to total protein (left panel). EGFRvIII cells transfected with either mismatch
siRNA or Elk-1 siRNA were analyzed by immunoblot analysis using antibodies against Elk-1, and -actin
(right panel).
51
4.1.3 EGFRvIII requires a putative AP-1 response element to transcriptionally
regulate TBP
The requirement of the putative AP-1 response element for EGFRvIII
mediated regulation of TBP promoter activity was addressed via site-directed
mutagenesis of the AP-1 site only, as previously described and detailed in the
Materials and Methods (Figure 15A). In context to the -736/+66 and -230/+66
promoter fragments, the AP-1 mutant promoter activity was not responsive to
AG1478 treatment in the U87-EGFRvIII cells (Figure 15B, Left). The activity of
this construct was compared to the activity of the wild type and ETS mutant
constructs in these cells. The AP-1 mutant promoter activity was substantially
lower in the U87-EGFRvIII compared to the activity of the wild type and ETS
mutant promoters (Figure 15B, Right). This suggests that EGFRvIII requires the
AP-1 response element to induce TBP promoter activity.
4.1.4 EGFRvIII directly targets c-Jun and c-Fos to the TBP promoter
To identify the transcription factors targeted to the TBP promoter by
EGFRvIII, we examined c-Fos and c-Jun, two members of the AP-1 transcription
factor family. The effect of modulating c-Jun and c-Fos expression levels on
TBP promoter activity in EGFRvIII expressing cells was first addressed.
Immunoblot analysis confirmed the siRNA-mediated decrease in c-Jun and c-Fos
protein levels (Figure 15C, Right). Decrease expression of c-Jun and/or c-Fos
inhibited EGFRvIII mediated induction of the TBP promoter (Figure 15C, Left).
This is in contrast to EGFR1, where no effect on the TBP promoter was observed
52
Fold change in TBP
promoter activity
0.0
0.5
1.0
1.5
- +
mm siRNA:
- -
c-Fos siRNA:
+ - - +
c-Jun siRNA:
+ - - +
U87-EGFRvII1
B.
C.
-736-AP1mut-Luc
-736-Etsmut-Luc
-736 TBP-Luc
-230-AP1mut-Luc
-230 TBP-Luc
c-Fos siRNA:
c-Fos
β-actin
+
-
U87-EGFRvIII
0.0
0.5
1.0
1.5
Fold change in TBP
promoter activity
U87-EGFRvIII
0.0
0.5
1.0
1.5
-
+
-
+ - + - +
Fold change in TBP
promoter activity
A.
Elk-1
AP-1
actggcggaagtgacattatcaacgcgcgccagggttcagtgaggtcgggca
-54
-1
hTBP-Luc Luciferase
-230 -84 -1 +66
Ets AP1
- hTBPAP1mut-Luc Luciferase
-230 -84 -1 +66
Ets AP1
-736
-736
AG1478:
c-Jun
β-actin
c-Jun siRNA:
+
-
Figure 15. EGFRvIII transcriptionally regulates TBP via an AP-1 response element. A).
Schematic of the annotated TBP promoter containing an adjacent AP-1 site (top). Schematic of
the wild type and AP-1 mutant TBP promoter reporter constructs used for promoter assays
(bottom). B). Cells were transfected with 2 ug of the designated promoter reporter construct and
serum starved the following day. 6-8 hours post-serum starvation cells were treated with AG1478
(10 uM) overnight. Cells were harvested the following day and analyzed for luciferase activity
and normalized to total protein (left panel). Equal amounts of lysates were analyzed for β-
galactosiodase activity and fold changes were calculated based on the wild type TBP promoter
activity. C). U87-EGFRvIII cells were transiently co-transfected with 2 ug of the wild type TBP
promoter reporter construct and either mismatch siRNA (-), c-Jun siRNA (+), and/or c-Fos siRNA
(+). Fold changes were calculated based on control (TBP promoter + mm siRNA) and normalized
to total protein (left panel). U87-EGFRvIII cells transfected with either mismatch siRNA or c-
Fos or c-Jun siRNA were analyzed for protein expression. Immunoblot analysis was performed
using lysates prepared from transfected cells using antibodies against c-Fos, c-Jun, and β-actin
(right panel).
53
D.
U87-EGFRvIII
+ + + - - - -
-3358/-3315 primers
TBP promoter occupancy
Elk-1 c-Jun c-Fos IgG
0
1
3
5
7
9
11
AG1478:
ChIP Antibody:
+ + + - - -
-119/+5 primers
TBP promoter occupancy
Elk-1 c-Jun c-Fos
0
1
3
5
7
9
11
Figure 15. cont. EGFRvIII transcriptionally regulates TBP via a AP-1 response element. D).
ChIP assays were performed using previously described primer sets on serum starved U87-
EGFRvIII cells treated with AG1478 (10 uM) overnight where designated. qPCR was performed
to quantify the amplified DNA. The fold change in TBP occupancy was calculated based on
untreated control after normalization to input and PCR efficiency. The values shown are the mean
± SEM of at least 3 independent determinations.
54
as discussed in Chapter 3 (Figure 12C), further suggesting that EGFR1 and
EGFRvIII transcriptionally regulate TBP through distinct mechanisms.
To determine whether the AP-1 dependent effects on the TBP promoter were
direct or indirect, ChIP analysis was performed using U87-EGFRvIII cells with
and without AG1478 treatment. c-Jun and c-Fos were both significantly recruited
to the TBP promoter and treatment with AG1478 resulted in a significant loss of
their occupancy (Figure 15D, Left). In contrast, there was no EGFRvIII specific
recruitment of Elk-1 observed (Figure 15D, Left). The upstream primers
(-3358/-3315) efficiently amplified input chromatin but occupancy of Elk-1, c-
Fos, or c-Jun was undetectable confirming the specificity of the recruitment of
these transcription factors to select sites within the TBP promoter (Figure 15D,
Right).
4.2 SUMMARY
In this chapter, it was confirmed that EGFRvIII regulates TBP promoter
activity through a different signal transduction pathway than EGFR1 by targeting
c-Jun and c-Fos to the TBP promoter.
4.3 MATERIALS AND METHODS
4.3.1 Cell Culture
All NR6, NIH3T3, and U87 cell lines were described in Chapter 2. All
cell lines were grown at 37 C and 5% CO2.
55
4.3.2 Western Blot Analysis
Cells were seeded into a 10 cm dish so that they were 85-90% confluent
on the day of isolation. Chemical treatments and isolation of lysates was
described in Chapter 2. Samples were prepared for SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) and immunoblot analysis as described in Chapter 2.
Antibodies for c-Fos, c-Jun, Elk-1, JNK1/JNK2, IgG, TBP, β-actin were as
described in Chapter 3. Goat Anti-Mouse IgG Peroxidase conjugated or Goat
Anti-Rabbit IgG peroxidase conjugated secondary antibodies and
chemiluminescence reagent were described in Chapter 2.
4.3.3 lasmid DNAs
All human wild type and ETS mutant TBP promoter-luciferase constructs
were a gift from Dr. Diane Hawley (16) except for mutations made in the AP-1
response element, which is described under site-directed mutagenesis. The
human CMV-β-galactosidase reporter gene construct was a gift from Dr. Amy
Lee. The human c-Fos-luciferase promoter construct was provided by Dr. Craig
Hauser (17).
4.3.4 Transient Transfections
Cell plating, transfection, AG1478 treatment conditions, and lysate
isolation were described in Chapter 2. Where designated cells where transfected
with .1 ug of CMV- βgal reporter plasmid. Where designated cells were treated
with p38 inhibitor (Calbiochem # SB220025), JNK inhibitor V (Calbiochem #
56
420129), MEK inhibitor ( Promega # U0126) overnight and then isolated as
previously described.
The protocol for transient transfections of synthesized human small
interfering RNA (siRNA) oligonucleotide sequences and/or reporter plasmids was
described in Chapter 3.
4.3.5 Preparation of Cell lysates for Protein, Luciferase and β-gal Assays
The protein and luciferase assay protocol was described in Chapter 2.
Where designated, β-gal assays were performed in a 96-well plate to measure
transfection efficiency. β-gal activity was determined as described in Chapter 3.
4.3.6 Site-directed mutagenesis
Site-directed mutagenesis of the -736/+66, -230/+66, and -4500/+66 TBP
promoter-luciferase constructs were performed following Stratagene’s protocol
for QuickChange Site-Directed Mutagenesis Kit (cat # 200518, or 200521-
QuickChange II XL Site-Directed Mutagenensis Kit). Primers used for the ETS
mutation (G-47A) and the AP-1 mutation (GTGATGTTAT) were described in
Chapter 3.
4.3.7 Real time PCR
Real-time PCR was performed using SYBR Green Supermix (Biorad #
170-8882) on a MX3000P system (Stratagene) as described in Chapter 3.
Calculations and TBP primer sequences were described in Chapter 3.
57
4.3.8 Chromatin Immunoprecipitation Assay
Chromatin immunoprecipitation (ChIP) assays were performed as described
in (62) and in Chapter 3, except no EGF treatment.
58
CHAPTER 5: Internalization distinguishes EGFR1 and EGFRvIII signaling
We have discovered a novel mechanism for regulating TBP which
includes the activation of c-Jun and c-Fos. This alternative mechanism could be a
result of a number of different issues. RTK mediated signaling cascades are
influenced by multiple factors, for example, ligand and receptor concentration,
rate of receptor internalization and recycling, and degradation of RTKs.
Endocytosis is a process where RTKs are internalized via clathrin coated pits
followed by entrance into the early endosome and where they either get degraded
in the lysosome or get recycled back to the plasma membrane. The transient
signaling nature of EGF-activated EGFR1 is aided via endocytosis. Different
ligands, however, result in differential temporal activation of endocytosis which
influences downstream signal transduction. This is one way ligands initiate
differential signal transduction pathways. We addressed the consequences of
prolonged presence of EGFR1 at the cell surface, thus mimicking EGFRvIII,
which is not rapidly internalized and recycled like EGFR1.
5.1 RESULTS
5.1.1 Overexpression of c-Jun and c-Fos regulates TBP promoter activity
We first analyzed the expression levels of Elk-1, c-Jun, and c-Fos in the
U87-EGFR1 and the U87-EGFRvIII cell lines. No significant expression
differences of these proteins in EGFR1 or EGFRvIII cell lines were observed that
might account for the differential requirement of Elk-1 versus AP-1 for receptor-
mediated induction of TBP expression (Figure 16A). However, the EGFRvIII
59
expressing cells had slightly higher c-Jun protein levels. As expected, based on
previous studies, inhibition of receptor activation decreased the expression levels
of c-Jun. Although EGFR1 and EGFRvIII have been shown to regulate c-Fos
promoter activity, under these conditions, c-Fos protein levels were insensitive to
AG1478. We next tested whether we could force TBP promoter induction in the
U87, U87-EGFR1, and U87-EGFRvIII cells by overexpressing c-Jun and/or c-
Fos. Overexpression of either c-Jun or c-Fos did not induce TBP promoter
activity in U87 (Figure 16B). Overexpression of c-Jun or c-Fos in the U87-
EGFR1 and U87-EGFRvIII cell lines showed a very modest dose dependent
increase in TBP promoter activity. Overexpression of both c-Jun and c-Fos
however resulted in a synergistic increase in TBP promoter activity in a serum
dependent manner in either the U87 or U87-EGFR1 cells (Figure 16C).
Overexpression of both c-Jun and c-Fos in the U87-EGFRvIII cells, however,
resulted in a less dramatic dose dependent increase in TBP promoter activity.
5.1.2 The rate of EGFR1 internalization influences the activation of downstream
signaling cascades
Recepter-mediated endocytosis is one way a cell regulates responses to
extracellular signals and this process entails internalization of ligand bound
receptors followed by either recycling of the receptor back to the cell surface or
degradation in the lysosome. Internalization of EGFR1 is a rapid process after
binding EGF. EGFRvIII on the other hand does not bind EGF and does not
activate this rapid internalization process (26). We asked whether inhibiting
60
U87-
EGFR1
U87-
EGFRvIII
c-Fos
c-Jun
β-actin
AG1478: - + - +
Elk-1
B.
c-Jun:
c-Fos:
Fold change in TBP
promoter activity
U87
0 .125 .5 1
.125 .5 1 - - - -
- - -
0.0
0.5
1.0
1.5
A.
C. D.
0.0
0.5
1.5
2.5
U87-EGFRvIII
c-Fos: .125 .5 1
.125 .5 c-Jun: 0
- - - -
1 - - -
Fold change in TBP
promoter activity
U87-EGFR1
0.0
0.5
1.5
2.5
c-Fos: .125 .5 1
.125 .5 c-Jun: 0
- - - -
1 - - -
Fold change in TBP
promoter activity
E.
U87
cFos:
cJun:
0
5
10
15
20
cFos:
cJun:
U87-EGFR1
0
5
10
15
20
cFos:
cJun: 0
0
.125
.125
.5
.5
1
1
U87-EGFRvIII
0
5
10
15
20
Fold change in TBP
promoter activity
Fold change in TBP
promoter activity
Fold change in TBP
promoter activity
0
0
.125
.125
.5
.5
1
1
0
0
.125
.125
.5
.5
1
1
Figure 16. Activated AP-1 complexes can regulate the TBP promoter. A). Immunoblot analysis
of WCL derived from U87-EGFR1 and U87-EGFRvIII cells treated with AG1478 (10 uM)
overnight. Elk-1, c-Jun, c-Fos, and β-actin antibodies were used for immunoblot analysis. B). In
U87 parental cells, increasing amounts of c-Jun or c-Fos expression vectors were transiently co-
expressed with the wild type TBP promoter. Two days after transfection, logarithmically growing
cells were isolated and cell lysates were generated and analyzed for luciferase activity, normalized
to total protein. C). U87-EGFR1 cells were treated as described in “B”. D). U87-EGFRvIII cells
were treated as described in “B”. E). In U87, U87-EGFR1, and U87-EGFRvIII cells, increasing
amounts of c-Jun and c-Fos expression vectors were transiently co-expressed with the wild type
TBP promoter. Two days after transfection, logarithmically growing cells were isolated and cell
lysates were generated and analyzed for luciferase activity, normalized to total protein. The fold
change in TBP promoter activity was calculated based on control (TBP promoter + empty vector).
The values shown are the mean ± SEM of at least 2 independent determinations.
61
internalization and consequently recycling of EGFR1 affected the requirement of
Elk-1 for EGFR1 to regulate TBP gene expression. EGFR1-mediated endocytosis
is a clathrin coated pit-dependent process which can be inhibited by diminishing
clathrin expression (54). Therefore, we asked if prolonged EGFR1 cell membrane
occupancy switched the requirement of Elk-1 to AP-1, thus mimicking
downstream EGFRvIII signaling. The ETS mutant TBP promoter activity
displayed little sensitivity to AG1478 treatment under normal endocytic processes
conditions (Figure 17A). However, prolonged EGFR1 occupancy at the plasma
membrane resulted in the ETS mutant TBP promoter becoming markedly
activated and sensitive to AG1478 (Figure 17A). If EGFR1-mediated endocytosis
is a major factor in the dependence of Elk-1 versus AP-1 on TBP promoter
activity, we should observe a requirement for the AP-1 regulatory element and a
loss for the requirement of the Elk-1 regulatory element with prolonged cell
membrane occupancy of EGFR1. When EGFR1 undergoes its normal
internalization process, the AP-1 mutant promoter is significantly stimulated and
sensitive to AG1478 treatment (Figure 17A). However, decreased clathrin
expression thus prolonged EGFR1 cell surface occupancy impaired AP-1 mutant
promoter activity rendering the promoter insensitive to AG1478 treatment (Figure
17A). Simultaneous mutation of both the Elk-1 and AP-1 sites within the TBP
promoter, resulted in promoter activity that was was unresponsive under all
conditions (Figure 17A).
62
0.0
0.5
1.0
1.5
2.0
U87-EGFRvIII
A.
B.
AG1478:
Clathrin siRNA:
+
+ -
-
-
+ +
+ -
-
-
+
Fold change in promoter activity
Clathrin
β-actin
Clathrin siRNA:
+
-
+
-
C.
0
1
2
3
4
5
6
7
8
+
+ -
-
-
+
+
+
-
-
-
+
+
+
+
-
- -
-
+
-
+
-
+
AG1478:
EGF:
+ + + + + + + + + + + +
Clathrin siRNA:
TBP-ETSmutant-Luc
TBP-AP1mutant-Luc
TBP-ETS/AP1mutant-luc
Normalized promoter
activity *10
-3
Figure 17. Inhibition of EGFR1 internalization mimics EGFRvIII signaling. A). Logarithmically
growing U87-EGFR1 cells were transfected with 100 nM clathrin siRNA. Two days later, cells
were split and transfected with the designated TBP promoter plasmid and clathrin siRNA. Two
days later cells were serum deprived and 6-8 hours later treated with EGF (2 or 50 ng/ml) and
where designated, AG1478 (10 uM) overnight. The next day, cell lysates were generated and
analyzed for luciferase activity, normalized to protein. B). Logarithmically growing U87-
EGFRvIII cells were transfected and treated as described in “A”. C). Immunoblot analysis on
lysates derived from cells transfected with clathrin siRNA. Antibodies against clathrin and actin
were used.
63
We next tested the effect of clathrin siRNA on EGFRvIII-mediated
regulation of TBP expression. Since this receptor is not internalized, modulation
of clathrin levels should not affect EGFRvIII-mediated effects on TBP promoter
activity. Consistent with this notion, decreased clathrin protein levels had no
affect on the ability of EGFRvIII to activate the ETS mutant TBP promoter
(Figure 17B). Immunoblot analysis confirmed a decrease in clathrin protein
levels in the presence of clathrin siRNAs (Figure 17C).
5.2 SUMMARY
Modulating the half-life of EGFR1 at the cell surface switches the
requirement of Elk-1 to AP-1 for TBP promoter induction. The inherent impaired
internalization of EGFRvIII results in prolonged signaling which differentiates it
from EGFR1-mediated signaling.
5.3 MATERIALS AND METHODS
5.3.1 Cell Culture
All U87 cell lines were described in Chapter 2. All cell lines were grown
at 37 C and 5% CO2.
5.3.2 Western Blot Analysis
Cells were seeded into a 10 cm dish so that they were 85-90% confluent
on the day of isolation. Chemical treatments and isolation of lysates was
described in Chapter 2. Samples were prepared for SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) and immunoblot analysis as described in Chapter 2.
64
Antibodies for clathrin heavy chain were purchased from BD Transduction Labs.
Goat Anti-Mouse IgG Peroxidase conjugated or Goat Anti-Rabbit IgG peroxidase
conjugated secondary antibodies and chemiluminescence reagent were described
in Chapter 2.
5.3.3 Plasmid DNAs
All human wild type and ETS mutant TBP promoter-luciferase constructs
were a gift from Dr. Diane Hawley (16) except for mutations made in the AP-1
response element, which is described under site-directed mutagenesis in Chapter
3.
5.3.4 Transient Transfections
Cell plating, transfection, AG1478 treatment conditions, and lysate
isolation were described in Chapter 2. Where designated cells where transfected
with .1 ug of CMV- βgal reporter plasmid. The protocol for transient
transfections of synthesized human small interfering RNA (siRNA)
oligonucleotide sequences and/or reporter plasmids was described in Chapter 3,
except that an additional clathrin siRNA transfection took place 2 days prior to the
plasmid/siRNA double transfection as previously described. Clathrin heavy chain
siRNAs were purchased from Dharmacon (# D-004001-02-0005).
5.3.5 Preparation of Cell lysates for Protein, Luciferase Assays
The protein and luciferase assay protocol was described in Chapter 2.
65
CHAPTER 6: Discussion
6.1 Distinguishing the distinct gene targets of the EGFRs is of biological relevance
This study focused on understanding how oncogenic signal transduction
pathways regulate the expression of the central eukaryotic transcription initiation
factor, TBP. We investigated potential receptors and their downstream molecular
events involved in regulating the expression of TBP. Alterations in TBP
expression can promote cellular transformation via specific TBP-mediated
changes in the expression of select genes (28, 29). Our work supports that TBP
has proto-oncogenic properties, so we set out to identify what environmental cues
regulate the expression of TBP. We specifically investigated a potent mitogen,
EGF that might regulate TBP gene expression. As activation of EGFR1 via EGF
increases TBP expression in a mouse cell line, JB6, (64) we further revealed the
details of this mechanism and explored the regulation of TBP expression by other
structurally related EGFRs. Identifying the specific downstream gene targets that
distinguish EGFR1, EGFRvIII, and HER2 signaling is of importance because
these receptors are major contributing factors in a variety of aggressive cancers
(25). Defining EGFR1, EGFRvIII, or HER2 specific signaling is complicated by
co-expression of multiple EGFR family members. These receptors can
heterodimerize with one another to diversify their signaling, thereby obscuring the
role of each receptor. However, cellular proliferation is strongly regulated by
EGFR-Ras signaling and cell survival is strongly regulated by HER2-PI3K
signaling (25). Regulation of cellular proliferation and cell survival are the
66
foundations in the development of cancer and EGFR1 and HER2 can transform
cells independent of the expression of other EGFRs (1, 4). Distinguishing the
distinct signal transduction pathways of EGFR1 and HER2 will enhance our
understanding of the complex mechanisms involved in gene regulation that lead
to their transforming properties. Our approach has led to distinguishing functions
for EGFR1, EGFRvIII, and HER2 specific signaling by using receptor-specific
transformed cell lines.
6.2 EGFR1, but not HER2, regulates TBP gene expression
Both EGFR1 and HER2 activate Ras and PI3K mediated signaling
pathways to regulate distinct and overlapping targets, such as, c-Jun, c-Fos,
NFkB, and Ets factors (33). Our work, however, supports that EGFR1 regulates
TBP gene expression in an Ets dependent manner, whereas, HER2 does not
regulate TBP expression. This is presumably achieved via differences in their
intracellular receptor domains, mode of activation, ligand concentration, receptor
dimer composition, or differences in internalization and recycling properties.
Unlike EGFR1, HER2 undergoes prolonged signaling due to delayed endocytosis
and increased recycling, and also unlike EGFR1, HER2’s major signaling target is
PI3K (6, 25). Since we have shown that TBP is regulated via EGF-MAPK
pathway and not via PI3K pathway, this may explain HER2’s inability to regulate
TBP expression (30). Alternatively, EGFR1 or HER2 may promote differential
local populations of specific effectors, which can shift shared upstream signals
into alternative downstream signals. The inherent differences that exist between
67
EGFR1 and HER2 allow activation of a specific combination of molecules that
dictate the desired biological outcome, and in this case, TBP is a gene target of
EGFR1 but not HER2 signaling. Since most EGFR1 or HER2-associated cancers
overexpress EGFR1 or HER2, it is important to further define their receptor-
specific signaling.
6.3 EGFR1 requires Elk-1 to regulate TBP gene expression
The EGF-MAPK-TBP regulatory pathway was further detailed to identify
the specific Ets factor and MAPK isoforms involved. Out of the 30 different Ets
factors, only 6 are ubiquitously expressed. Of these six, only Elk-1 requires the
activation of all three MAPK classes (61). Since induction of TBP expression
requires the simultaneous activation of the 3 classes of MAPKs we reasoned that
Elk-1 might be the factor that regulates TBP promoter activity. We unveiled that
EGFR1 regulates the TBP promoter via an Elk-1 response element (64). Elk-1
recruitment to DNA requires phosphorylation at specific serine and threonine
residues in order to efficiently bind promoter regions of target genes and make
specific protein-protein interactions with its binding partners (61). Each MAPK
class targets specific domains within Elk-1 to modify Elk-1 in such a way that it
binds to and allows for activation of the TBP promoter.
6.4 JNK1 positively regulates, whereas JNK2 negatively regulates the
phosphorylation state of Elk-1
We began identifying the specific roles of the different isoforms of the
MAPKs involved in regulating TBP expression, starting with the JNKs. There are
68
three isoforms of JNK; JNK1, JNK2, and JNK3 whose direct targets have become
debatable (22). Our studies show that activated JNK1 cooperates with p38 and
ERK to simultaneously phosphorylate Elk-1 to increase the expression of TBP. In
MEFs, this results in increased c-Jun expression and cellular proliferation rates.
Our studies also show that JNK2 serves to negatively regulate TBP expression by
constitutively preventing Elk-1 phosphorylation, resulting in a decrease in its
recruitment to the TBP promoter (63). Upon activation the JNKs have been
shown to activate specific cellular targets, such as Elk-1, c-Jun, and JunD, by
phosphorylating specific serine and threonine residues within specific domains
recognized in the target proteins, at least in vitro. However we showed, JNK2
mediated decreases in the phosphorylation state of Elk-1, in vivo (63). This
occurs through an unknown mechanism, either by enhancing the action of a
phosphatase, or alternatively, JNK2 may block JNK1-mediated phosophorylation
of Elk-1 to sterically hinder its activation. This is not a novel mechanism in
regulating proto-oncogenes, however. c-Jun, for example, is kept constitutively
inactive in resting cells by GSK3 and casein kinase II (5). On the other hand, this
is a novel mechanism for negatively regulating TBP expression. This suggests
that a critical balance between JNK1 and JNK2 expression and/or activation is
important for regulating TBP expression. Given that the JNKs are known to
regulate cellular proliferation, apoptosis, survival, promote transformation, and
are important regulators of transcription, defining the role of each isoform is
important (15, 34). Our studies support the idea that JNK1 may promote
69
oncogenesis, whereas JNK2 may function as a tumor suppressor, in part, by their
ability to regulate the expression of TBP.
6.5 EGFR1 and EGFRvIII differentially regulate TBP promoter activity
Despite the sequence similarities between EGFR1 and EGFRvIII, our
work shows that unique signaling exists between these two receptors. EGFRvIII
regulates TBP promoter activity through the direct recruitment of c-Jun and c-Fos
transcription factors, not Elk-1. This may be a result of differences in how
EGFR1 and EGFRvIII are activated or regulated. EGFR1 and EGFRvIII may
differentialy activate different isoforms of p38 or ERK, which can lead to distinct
downstream signal transduction pathways.
Signaling pathways can be influenced by local receptor and/or ligand
concentration. The requirement of Elk-1 for EGFR1-mediated induction of the
TBP promoter is independent of receptor concentration since both Huh-7,
representing moderate expression, and U87-EGFR1, representing a high level of
receptor, were both Elk-1 dependent. These studies demonstrate that TBP is a
bonafide target of EGFR1 signaling.
The difference in regulatory pathways between EGFR1 and EGFRvIII
may be a result of the constitutive activity of EGFRvIII. Sustained verses
transient activation of ERK can lead to the activation of distinct signaling
pathways. The sustained signaling of EGFRvIII therefore may result in the
activation of alternative downstream ERK signaling pathways that induce specific
genes in order to enhance the oncogenic properties of the cells. In the Chinese
70
hamster lung fibroblast cell line, CCI39, for example, sustained ERK activation is
required for enhanced c-Jun expression (2). It is also possible that the sustained
signaling of EGFRvIII activates alternative effectors such as PP2b, or a similar
phosphatase(s), resulting in dephosphorylation of Elk-1. Protein phosphatase 2b
(PP2b) dephosphorylates EGF-activated Elk-1 to inhibit its transactivation
potential and PP2b enhances the transactivation potential of c-Jun (5, 52).
EGFRvIII activation, therefore, might not adequately phosphorylate Elk-1 such
that it is unable to be recruited to the TBP promoter. Although speculative, there
also exists the possibility that EGFRvIII differentially activates the JNKs in such
a way that its major target is c-Jun not Elk-1. Another EGF-Ras downstream
kinase that negatively regulates Elk-1 phosphorylation and activation is KSR,
kinase suppressor of ras, which may also be involved (52). The ability of TBP to
be regulated by this AP-1 consensus site uncovers a new regulatory mechanism
for TBP that may play a role in the disease state or during development. The
AP-1 site within the TBP promoter can be targeted in non-EGFRvIII expressing
cells by ectopically overexpressing c-Jun and c-Fos. This suggests that EGFRvIII
signaling provides novel regulatory mechanism for modulating the expression of
the TBP gene in an oncogenic state.
6.6 A composite ETS/AP-1 site within the TBP promoter allows for differential
regulation by EGFR1 and EGFRvIII
We are the first to identify distinct transcription factors targeted by EGFR1
and EGFRvIII that converge to transcriptionally regulate the same gene, TBP.
71
Several Ras dependent genes, such as, Heparin-Binding-EGF, c-Fos, MMP-9, and
Collagenase contain an oncogene responsive element (ORE) comprised of
overlapping or nearby ETS and AP-1 elements which enhances the promoter’s
transcriptional activity (17, 35). It is possible then that EGFR1 and EGFRvIII
work together to exploit the composite ETS/AP-1 response elements within the
TBP promoter and other genes to ensure activation of these genes under multiple
conditions, as commonly found with targets downstream of oncogenic Ras. The
composite ETS/AP-1 sites may allow TBP expression to be induced through
activation of both transient and sustained signaling pathways. We propose that
EGFR1 and EGFRvIII cooperatively regulate the transcription of TBP by
individually targeting distinct response elements to progress tumors to the late
stage aggressive state, as amplification of EGFR1 amplification precedes
EGFRvIII expression (42, 51).
6.7 Endocytosis plays a key role in distinguishing EGFR1 and EGFRvIII mediated
signal transduction
Receptor cell surface half-life plays a major role in implementing transient
EGFR1 signaling for activation of the appropriate signaling pathway. When
EGF-activated EGFR1 internalization and recycling is inhibited, AP-1, not Elk-1,
is required. EGFR1 is rapidly internalized via clathrin coated pits after EGF
stimulation, whereas EGFRvIII escapes internalization. EGFRvIII undergoes
delayed endocytosis and degradation in such a way that at steady state equilibrium
active EGFRvIII at the plasma membrane is persistent (12). EGFR1 on the other
72
hand has multiple endocytic ‘signals’ that are activated in its intracellular domain
to mediate its signaling intensity, whereas EGFRvIII has impaired activation of
these sites, thus allowing it to escape endocytosis. Persistent EGFR1 at the
plasma membrane via decreased clathrin expression, mimicked EGFRvIII
signaling to the TBP promoter. This suggests that the unattenuated signaling by
EGFRvIII is a major catalyst causing it to signal through AP-1. The cell therefore
has another layer of regulation through receptor and ligand-dependent
internalization and recycling rates to regulate gene expression.
The regulation of gene expression via signal transduction cascades are also
influenced significantly by the micro-environment (pH, ligand availability, etc).
EGFRvIII may provide cancer cells with alternative signaling pathways that
cooperate with EGFR1 in a paracrine fashion to enhance the transformation
potential.
These studies reveal novel mechanisms for regulating TBP expression and
novel regulatory mechanisms for EGFR1 and EGFRvIII. The correlation of these
mechanisms on other genes with similar promoter elements will be interesting to
unfold.
73
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81
Abstract (if available)
Abstract
The Epidermal Growth Factor Receptor (EGFR) family regulates essential biological processes upon receptor activation and deregulation of these receptors is detrimental to cellular homeostasis. HER2 and EGFR1 are two members of the EGFR receptor tyrosine kinase family. Several human cancers are linked to either HER2 or EGFR1 overexpression or expression of EGFR1 variants, like EGFRvIII. One important gene target of EGF-mediated signaling is the TATA-binding protein (TBP) (64). TBP is a central eukaryotic transcription initiation factor required by all nuclear RNA polymerases for transcription. Selective changes in gene expression patterns occur when TBP expression levels are altered (8, 57) and increased expression of TBP has been shown to promote cellular transformation (28). This study focuses on if and how activated HER2, EGFR1, and EGFRvIII regulate TBP expression. Here we show that EGFR1 and EGFRvIII, but not HER2, transcriptionally regulate TBP expression. EGFR1 regulates the TBP promoter via the recruitment of Elk-1 and EGFRvIII regulates the TBP promoter via recruitment of c-Jun and c-Fos. Furthermore, EGFR1 and EGFRvIII-mediated regulation of TBP expression is differentiated by transient versus sustained receptor activation, which is an effect of differential receptor internalization and recycling rates. Aside from the significance associated with understanding the mechanism by which this integral transcription factor is regulated in a transformed environment, the identity of specific downstream targets distinguishing the EGFRs is of clinical significance.
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Asset Metadata
Creator
Fromm, Jody Arlene
(author)
Core Title
Mechanisms for regulating the expression of the TATA-binding protein
School
Keck School of Medicine
Degree
Doctor of Philosophy
Degree Program
Biochemistry and Molecular Biology
Degree Conferral Date
2007-08
Publication Date
07/03/2009
Defense Date
06/08/2007
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
AP-1,EGFR,Elk-1,gene regulation,OAI-PMH Harvest,oncogenes,TBP,transcriptional regulation
Language
English
Advisor
Johnson, Deborah L. (
committee chair
), Frenkel, Baruch (
committee member
), Garner, Judy A. (
committee member
)
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jodyfromm@gmail.com
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https://doi.org/10.25549/usctheses-m573
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Fromm, Jody Arlene
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(contributing entity),
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cisadmin@lib.usc.edu
Tags
AP-1
EGFR
Elk-1
gene regulation
oncogenes
TBP
transcriptional regulation