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1. 中文摘要
細胞受到生長因子、賀爾蒙及其他刺激物的刺激，會經由細胞內訊息傳遞
來影響某些特殊基因的表現，以達成細胞功能的發揮。基於過去三年的研究成
果，我們在第四年計畫的每一個分項計畫，將把研究焦點更為集中來執行。第
一分項乃基於過去我們對 Sp1 在基因轉錄調控得知其可扮演一個 anchor 蛋白的
角色，可把其他轉錄因子如 c-Jun 及 VDR 帶至基因 promoter，因此在本年度將
把研究重點放在 c-Jun、VDR 及 Sp1 轉譯之後的蛋白修飾，特別針對磷酸化及乙
醯化，探討其如何影響基因表現的機制。在第二分項我們將集中在癌細胞
Met/Ron 及 EGFR/Neu 之訊息路徑研究的探討，特別將把焦點放在 Stat3 及 Eps8
媒介癌化相關基因表現的調控機制。在第三分項計畫則著眼探討細胞內抗細胞
凋亡的機制及其訊息傳遞。本年度將繼續第三年的成果，把焦點放在探討
Fas/Fas-L 互相作用之功能，細胞培養基質僵硬度影響細胞生長及鋰其抗細胞凋
亡的訊息傳遞機制。
配合各分項計畫的推動需求，在過去三年期間，我們在成大已建立一個研
究細胞訊息傳遞的核心設施，包括蛋白質體學、光學儀器、基因微陣列分析等
設施，以期協助計畫成員追求學術研究的卓越。
1. Executive Summary
Gene expression is regulated through intracellular signal transduction upon the
stimulation of growth factors, hormones and other stimulants. There are three
subprojects in this proposal. Based on the past three years research results, we make
more focus on our research directions in each subproject in the fourth year of this
proposal. In Sub-Project (I), we focus on the novel function of Sp1 that could serve as
an anchor protein to recruit other transcription factors to the gene promoter in the
regulation of gene expression. The functional role of post-translational modification
(phosphorylation and acetylation) of transcription factors c-Jun, VDR and Sp1 in the
transcriptional regulation of cellular genes is our major research direction. In
Sub-Project (II), we have integrated the whole subproject to study the signaling
pathways of receptor tyrosine kinases in human cancer. Since Met/Ron and
EGFR/Neu are involved in many human cancers including bladder cancer, colorectal
cancer, lung adenoma and liver cancers, we focus on these receptor tyrosine kinases
and the downstream potential signaling molecules, particularly Eps8 and Stat3, which
participate in these four kinds of human cancers. In Sub-Project (III), novel signal
transduction mechanisms that mediate anti-apoptotic effects in patho-biology.
Following the third year results, we will focus on the mechanistic studies regarding to
interaction of Fas/Fas-L, substratum rigidity- controlled cell behaviors such as
regulation of focal adhesion protein and cell survival, and conteractions of lithium to
ceramide-induced cell death.
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We have established the core laboratory facilities at NCKU, in the past three
years, which are essential for the success of the project. Deployment of larger
instruments such as mass spectrometers, confocal microscope, fluorescence and
chemiluminescence analyzers, together with biochips allow the researchers involved
in this project to compete internationally and pursuit the research excellency.
2. General Description
The normal cellular function is under a sophisticated regulation network, so
called “signal transduction”, to support the integrity of the system. When cellular
growth control is abnormal, for example, the cell continuously grows until a tumor is
formed which may damage the neighboring tissue and cause the organism to die. In
addition, when a cell should go to apoptosis but does not, its presence may block the
function of the neighboring cells and the whole tissue. Thus, to continuously perform
normal cellular function, a cell needs to be cooperatively regulated by thousands of
signal transduction processes within itself. Furthermore, the signal transmission is
dynamic and cross-talk may occur within the cell. Therefore, it is also necessary for
scientists to work with cross-talk in the research field of signal transduction. We
propose this project to integrate into a single research team all the intelligent scientists
working in this field in southern Taiwan. So far, our team has been involved
extensively in signal transduction and gene regulation research and has provided
major contributions to the field. Among them only two of the more significant
discoveries will be mentioned here. First, in the study of how c-Jun and Sp1 work
cooperatively in the activation of 12(S)-lipoxygenase expression, we discovered a
novel function of Sp1 as a carrier to bring the transcription factor c-Jun to the GC-rich
box-containing gene promoter. This is amongst the first few discoveries of such a
novel transcriptional factor function. Second, in the studies of HBV-related
hepato-carcinogenesis, we found that the mutated pre-S proteins of the hepatitis B
viral surface antigen are commonly present in liver tissues of chronic hepatitis B viral
infection, and the pre-S mutants may result in the down-regulation of small HbsAg in
endoplasmic reticulum (ER) resulting in ER stress. Through intimate contact and
intergration in this project, we will contribute to address, at the molecular level, the
tumorigenesis of the most important cancers in Taiwan. Also, we will be able to
provide knowledge about the regulation of transcriptional factors in mediating gene
expression and signal transduction in growth and apoptosis control. We have divided
this proposal, into three sub-proposals; (I) functional interaction of transcription
factors in gene expression; (II) receptor tyrosine kinases signaling through Stat3/Eps8
in human cancer; and (III) studies of signal transduction mechanisms that contribute
to tumor cell survival.
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3. Objectives
Specifically, our aims, which will be actualized by three subprojects, are to:
1) Elucidate functional interactions of transcription factors in gene expression
regulation;
2) Study receptor tyrosine kinases signaling through Stat3/Eps8 in human cancer;
and
3) Elucidate novel signal transduction mechanisms that mediate apoptosis or
anti-apoptotic effects in patho-biology.
4. Interface and Integration between Overall and Sub-Projects
The study of cellular signaling pathways and gene regulation is our main shaft in
this project. Instead of looking at individual signaling pathways (single dimensional
studies), we conduct our studies from a multi-dimentional prospective. Through the
study of “new mechanism”, in
search of “new genes”,
hopefully we will discover
“new
functions”
in
Sub-Projects. In order to
improve
the
research
infrastructure in the NCKU
medical research center and
form a technical support base
for the whole project, we have
established six core laboratories
in Overall project. They are (1) Mass Spectrometry (2) Microscopic Facility (3)
Inducible Gene Expression (4) Functional Genomics and (5) Structural Biology. The
interface between Overall and Sub-Projects is indicated in the following scheme.
5. Project Management
Dr. W.C.Chang is responsible for the project management. In order to achieve
our goals, the following strategies will be reinforced.
1) Integration: There will be frequent intra-subproject interactions. Inter-project
meetings will also be held regularly to monitor our progress and help to solve
problems. With careful supervision by the principal investigators, team spirit is
one of top priority that must be achieved early on in the project. Moreover, any
novel molecules, transcriptional factors or signal transduction pathways found in
one sub-project will be supported quickly by researchers working in on other
projects in terms of technical help, insight sharing and discussion on possible
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relationship with their own projects.
2) Core facilities: The responsible investigators in charge of the core facilities are all
experts in a particular technique. All will provide not only passive support but
also to be actively involved in providing insights, recommendations for new
techniques, and suggestions for novel research possibilities.
3) Quality control: In order to guarantee success and minimize unnecessary waste of
efforts, we have invited four distinguished scientists, three from abroad and one
local scientist to form an External Advisor Committee to oversee our research
progress annually. They will be responsible for critical appraisal of our research
directions, results and give important recommendations. Besides that our principle
investigators will supervise our team on a very frequent basis; scheduled meetings
and progress reports will be held regularly to quickly spot difficulties and find
solutions.
4) International collaborations: We have established collaborations with the
Biosignal Research Center of Kobe University headed by Dr. Ushio Kikkawa to
study the effect of phosphorylation/dephosphorylation on the c-Jun/Sp1
interaction. Similar collaboration relationship is also established with Dr. Nelson
Fausto of the University of Washington to study the signal transduction of ER
stress. Both of them will be actively involved in project.
6. Describe in detail the approaches and methodologies to implement the
proposed research works
CORE FACILITY I: Proteomics Research Core Laboratory (PRCL)
(Responsible Investigator: Pao-Chi Liao)
Objective:
To provide the following services:
(1) Training courses for 2-D gel electrophoresis (2D-GE)
(2) 2-D gel electrophoresis
(3) Protein MW measurement/confirmation
(4) Protein identification by mass specgtrometry (MS)
Major instrumentations:
Five sets 2-D gel electrophoresis (one set with multiple-gel capability)
Applied Biosystems DE-PRO MALDI-TOF mass spectrometer
Finnigan LCQ liquid chromatography-mass spectrometer
Applied Biosystems QSTAR LC-MS with o-MALDI (funded by NSC)
Plans for year 2005
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(1) Continue to provide services listed above.
(2) Continue to improve the quality of the services provided by PRCL.
(3) Provide more training programs to core laboratory users.
CORE FACILITY II: Time-lapse video microscopy/Biological imaging systems
(Responsible investigator: Tzeng-Horng Leu/Meng-Ru Shen)
Objective:
The main purpose of this core is to provide instrumentation support of (1) time-lapse
video microscopy and (2) biological imaging systems for researchers in the MOE
Program for Promoting Academic Excellence of Universities.
Facilities and Equipments:
(1) Time-lapse video microscopy
Leica AS MDW system is purchased and set up for live cell imaging. “All
components like camera, shutters, piezo z-positoner and monochromator are fully
integrated and optimized for light efficiency and acquisition speed” in this system.
Even fast cell dynamics can be recorded in 4D. This instrument will provide recording
of intracellular proteins/organelle translocation as well as long-time observation of
cellular movement. The whole system started to provide service since the July of 2003.
The training course will be held two times for each year.
(2) Biological imaging systems
We have set up a core laboratory of optical imaging with the financial support from
MOE Program for Promoting Academic Excellence of Universities and Center for
Bioscience and Biotechnology, National Cheng Kung University. This core laboratory
is well equipped with (1) a new generation of confocal microscope for live cell
imaging system; (2) an atomic force microscope (AFM) coupled with a confocal laser
scanning biological microscope; (3) an inverted research microscope coupled with
high speed cooling CCD and fluorescence illuminators. The function of these set-ups
is to analyze the dynamic processes in living cells, such as cytoskeleton dynamics,
secretory membrane trafficking, cellular interactions, chromatin dynamics,
intracellular pH and calcium measurement with simultaneously electrophysilogical
recording. This core laboratory has provided service since January 2005. The
training courses will be held every 2 months.
CORE FACILITY III: Multiple inducible gene expression cell model laboratory
(Responsible Investigator: Hsiao-Sheng Liu)
Objective:
This core facility is of vital importance in gene regulation for individual subprojects.
In order to allow tightly regulated multiple gene expression, three regulated
expressions have been established: the lactose repressor system (Lac system)3, an
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insect hormone ecdysone-dependent expression system (Ecd system), and a
tetracycline-dependent expression system (Tet system). The first two are the inducible
systems using IPTG, and ponasterone A (ponA) as the inducers, respectively. The
latter is a repression system using tetracycline as the negative regulator. The objective
of this core facility is to assist PIs in each subproject to utilize these systems to
regulate the genes of interest.
Facility and service:
The mission of this core laboratory is to make the plasmids of various inducible
systems. In addition, it functions as consultant center to help each laboratory to
develop their inducible systems. GenePulser XcellTM (BioRad) is an electroporator,
which is extremely powerful for DNA, RNA and protein transferring with very high
efficiency.
CORE FACILITY Ⅳ: DNA Microarray
(Responsible Investigator: Hsiao-Sheng Liu)
We will produce investigator specified cDNA chips and conduct commercial as well
as custom-made chip microarray hybridization (cDNA and oligonucleotides) and data
analysis. Despite reviewer’s strong suggestion to process Affimetrix microarray
chips, however, we do not have the facilities (instruments as well as scanning and
analysis software specific for Affimetrix microarray chip analysis; cost about 8~10
million NT) to handle Affimetrix microarray chips. We are able to process other
oligonucleotide chips such us the chips from Agilent Technologies. The complete
gene list of all the customized chips and the updated information about this core
facility are posted and routinely maintained on the website (http://140.116.58.57). So
far, we have produced four versions of chips, and the chip 4 “oncogene and kinase”
chip is under active service now. Ras signal pathway chip is our version 5 chip and
will be released early this year. The chip 6 of cell cycle and apoptosis has been
designed, and will be released in year 2005, too. All the microarray data generated
via this core will be then integrated through established databases platform. As a
result, each principle investigator (P. I.) in the MOE will share their data with one
another and, if possible, the interested P. I. can utilize these data for further data
mining. As a part of MOE program, we will continue to bridge this top-notch
technique with the subprojects proposed in the MOE program. The ultimate goal of
this core facility is to assist all the P.I.s in achieving their excellence in the field of
signal transduction and function genomics. Ultimately, the overall research
environment in the NCKU medical center will be also accordingly upgraded.
This core will afford the following services:
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1. Commercial and custom-made chip microarray analysis (oligonucleotide and
cDNA formats)
2. Data analysis (gene annotation, cytoband analysis and advances DATA analysis)
3. Design and production of custom-made chip
CORE FACILITY Ⅴ: Structure Biology Core
A: Lab for NMR and Protein Expression
(Responsible Investigator: Woei-Jer Chuang)
The main purpose of this core is to provide instrumentation support and service to
MOE investigators.
The aims of this structural core lab are to:
(1) determine the 3D structures of proteins by NMR;
(2) produce large quantities of proteins for NMR studies; and
(3) predict protein structures by modeling and analyze proteins.
B: Division of Computer Stimulation and Peptides Synthesis (CF5-PS)
(Responsible Investigator: Wai-Ming Kan)
This core facility provides a peptide, phosphopeptide and small molecule and their
combinatorial library synthesis service for project investigators. Special focus is on
the application of these combinatorial libraries for the identification of probable
phosphorylation sequences. Establishment of methodology for parallel synthesis of
small molecules will be emphasized in the coming year, which provides tools for
further investigation or as “lead” discovery for anticancer agents related to this
project.
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Sub-project (I) Functional interaction of transcription factors in gene expression
(Principal Investigator: Wen-Chang Chang)
The ability of the core promoter to respond to activators is dependent on the
cooperative interaction between the transcription factors. Our previous studies
indicate a novel function of Sp1 that could serve as an anchor protein to recruit other
transcription factors c-Jun (Figure 1) and VDR to the promoter in cells. In this
Sub-project, we will focus our studies on the functional role of post-translational
modification of c-Jun, VDR and Sp1 in the transcriptional regulation of cellular genes.
The major specific aims of the third year of this Sub-project are as follows.
(1) Effect of PP2B-regulated dephosphorylation of c-Jun C-terminus on the
interaction between c-Jun and Sp1
(2) Deacetylation of Sp1 to regulate 12(S)-lipoxygenase transcription upon PMA
treatment in A431 cells
(3) Molecular mechanism of interaction between vitamin D receptor and Sp1 in gene
regulation
Figure 1. Sp1 functions as an anchor protein to recruit c-Jun to promoter of human
12(S)-lipoxygenase gene expression. (Chang, W.C., Prost. Other Lipid Mediat. 71,
277-285, 2003)
I-1-a: Functional mechanism of c-Jun/Sp1 interaction in the regulation of gene
transcription (Wen-Chang Chang and Ben-Kuen Chen)
We previously demonstrated that epidermal growth factor (EGF), transforming
growth factor , and phorbol 12-myristate 13-acetate (PMA) induce expression of
human 12(S)-lipoxygenase in human epidermoid carcinoma A431 cells (1-3) and that
EGF- and PMA-induced gene expression of 12(S)-lipoxygenase is regulated by the
9
functional interaction between c-Jun and Sp1 (4, 5). These studies also showed that
Sp1 can serve as an anchor protein to carry c-Jun to the promoter, and thus
transactivates the transcriptional activity of 12(S)-lipoxygenase gene (6). Furthermore,
we recently found that EGF-induced gene expression of keratin 16 is also regulated
by
c-Jun/Sp1
interaction
(7).
Because
PMA
induces
serine/threonine
dephosphorylation of c-Jun at the C-terminal domain (8), the functional role of this
dephosphorylation in regulating the c-Jun/Sp1 interaction was investigated. We found
that PMA induced dephosphorylation of c-Jun C-terminus in A431 cells. c-Jun mutant
TAM-67-M3A, which contains three substitute alanine residues at Thr-231, Ser-243,
and Ser-249, compared to TAM-67, bound more efficaciously with Sp1 and was about
twice as efficacious as TAM-67 in inhibiting the PMA-induced activity of the
12(S)-lipoxygenase promoter. Moreover, PP2B bound and dephosphorylated the
phospho-TAM-67. Inhibition of PP2B by using PP2B siRNA resulted in attenuating
the PMA-induced gene expression and c-Jun/Sp1 interaction. Our results indicated
that PP2B played an important role in regulating c-Jun/Sp1 interaction in
PMA-induced gene expression. In this study, we will analyze the PMA-induced
dephosphorylation sites of c-Jun C-terminus. The functional interaction between c-Jun
and PP2B will be also studied.
Experimental Design and Anticipated Results
Effect of PP2B-regulated dephosphorylation of c-Jun C-terminus on the
interaction between c-Jun and Sp1
Our preliminary data showed that PMA induced dephosphorylation of c-Jun
C-terminus. In order to identify the dephosphorylation sites of c-Jun C-terminus in
PMA-treated cells, specific dephosphorylation sites of c-Jun induced by PMA are
analyzed by stable isotope dimethyl labeling and mass spectrometry. The mutants of
c-Jun protein, TAM-67-T231A, TAM-67-S243A and TAM-67-S249A are constructed.
These mutants will also be expressed in cells and the interaction between Sp1 and
c-Jun mutants are analyzed by immunoprecipitation. Furthermore, the PMA-induced
dephosphorylation
anti-phospho-c-Jun
sites
T231
of
c-Jun
C-terminus
243
, anti-phosphoc-JunS
are
verified
and anti-phospho-c-Jun
S249
by
using
antibodies.
These studies will dissect the potential dephosphorylation sites of c-Jun protein
correlated to the binding to Sp1 in PMA-treated cells. In our studies, we have found
that c-Jun was targeted by PP2B in an in vitro phosphorylation and GST protein
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interaction assay. In order to study whether PP2B could bind to c-Jun in PMA-treated
cells, the GFP-c-Jun and RFP-PP2B are constructed. The colocalization of PP2B and
c-Jun will be analyzed by confocal microscopy in PMA-treated cells. The functional
interaction assay will also be performed by ChIP. To narrow down the interaction
domain of PP2B to c-Jun, 5`-truncated PP2B fusion protein, His-PP2B is constructed
and the interaction between c-Jun and truncated PP2B will be analyzed by protein
interaction assay. These results will help us to clarify the interaction domain of PP2B
to c-Jun and the functional interaction of PP2B/c-Jun in PMA-treated cells.
References
1.
Chang, W. C., Ning, C. C., Lin, M. T., and Huang, J. D. (1992). Epidermal
growth factor enhances a microsomal 12-lipoxygenase activity in A431 cells. J.
Biol. Chem. 267, 3657-3666.
2.
Chen, L. C., Chen, B. K., Liu, Y. W., and Chang, W. C. (1999). Induction of
12-lipoxygenase expression by transforming growth factor-alpha in human
epidermoid carcinoma A431 cells. FEBS Lett. 455, 105-110.
3.
Liu, Y. W., Asaoka, Y., Suzuki, H., Yoshimoto, T., Yamamoto, S., and Chang, W.
C. (1994). Induction of 12-lipoxygenase expression by epidermal growth factor
is mediated by protein kinase C in A431 cells. J. Pharmacol. Exp. Ther. 271,
567-573.
4.
Chen, B.K. and Chang, W.C. (2000) Functional interaction between c-Jun and
promoter factor Sp1 in epidermal growth factor-induced gene expression of
human 12(S)-lipoxygenase. Proc. Natl. Acad. Sci. USA. 97, 10406-10411.
5.
Chen, B. K., Tsai, T. Y., Huang, H. S., Chen, L. C., Chang, W. C., and Tsai, S. B.
(2002). Functional role of extracellular signal-regulated kinase activation and
c-Jun induction in phorbol ester-induced promoter activation of human
12(S)-lipoxygenase gene. J Biomed Sci 9, 156-165.
6.
Chang, W.C. (2003) Cell signaling and gene regulation of human 12(S)lipoxygenase expression. Prostaglandins Other Lipid Mediat. 71, 277-285.
7.
Wang, Y. N. and Chang, W. C. (2003) Induction of disease-associated keratin 16
gene expression by epidermal growth factor is regulated through cooperation of
transcription factor Sp1 and c-Jun. J Biol Chem. 278, 45848-45857.
8.
Boyle, W J., Smeal, T., Defize, L. H., Angel, P., Woodgett, J. R., Karin, M., and
Hunter, T. (1991) Activation of protein kinase C decreases phosphorylation of
11
c-Jun at sites that negatively regulate its DNA-binding activity. Cell 64, 573-584.
I-1-b: Deacetylation of Sp1 to regulate 12(S)-lipoxygenase transcription upon
PMA treatment in A431 cells (Jan-Jong Hung, Ben-Kuen Chen and
Wen-Chang Chang)
Sp1 is a basic transcriptional factor, which binds to the GC-rich region in the
promoter of target gene(s). It is involved in transcription of numerous genes by
recruiting other transcriptional factors to the promoters of target genes. Recent,
studies reveal that both of DNA binding ability and transactivational activity of Sp1
may be influenced by the post-translational modification of Sp1 such as
phosphorylation, glycosylation and acetylation (1, 2). Therefore, post-translational
modification on Sp1 due to interaction with other factors may play an important role
in regulation of Sp1 activity. Our previous studies show that transcription of
12(S)-lipoxygenase is regulated through interaction of c-Jun and Sp1 upon PMA or
EGF treatment in A431 cells. Sp1 may serve at least in part as a carrier to bring c-Jun
to the promoter, thus transactivating the transcriptional activity of human
12(S)-lipoxygenase gene (3-5). Recently, our preliminary data showed that Sp1 could
be acetylated in A431 cells, and the acetylation could be inhibited upon PMA
treatment. Furthermore, HDAC1, p300 recruited by Sp1 to promoter could be
increased under PMA treatment in A431 cells. We have also mapped that the Lys703
of Sp1 could be acetylated. The expression of 12(S)-Lipoxygenase was increased
under Sp1 (K703/A) overexpression in A431 cells. However, it is still unclear how
this interaction of c-Jun and Sp1 regulates transcription of its target gene(s). In this
study, we are interested in studying the post-translational modification of Sp1 to
regulate the transcription of 12(S)-lipoxygenase upon PMA treatment in A431 cells.
Experimental Design and Anticipated Results
Role of Sp1 acetylation on the interaction of c-Jun, HDAC1, p300 and Sp1
We have known that c-Jun can interacted with Sp1, and acetylation of Sp1 was
decreased under PMA treatment in A431 cells. It is unknown whether the acetylation
of Sp1 can affect the interaction of Sp1 and its interacted proteins such as c-Jun,
HDAC1 and p300. Therefore, plasmids, pBSSR-Sp1-His and pBSSR-Sp1
(K703/A)-His will be transfected into the A431 cells, and then cells will be treated
with PMA. Later, cells will be double stained with anti-His conjugated FITC and
anti-c-Jun, anti-HDAC1 or anti-p300 conjugated Cys5 to study the colocalization of
Sp1 with these proteins in vivo. In addition, the cell lysates will be used to do the
immunoprecipitation with anti-His and then analyze the interacted proteins with
immunoblot of anti-p300, anti-c-Jun and anti-HDAC1.
Role of
Sp1 upon TSA treatment on
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the transcription activity of
12(S)-lipoxygenase
Overexpression of Sp1 (K703/A) can induce the transcription of
12(S)-lipoxygenase in A431 cells. To confirm this result, cells will be treated with
TSA and then study the mRNA and protein level of 12(S)-lipoxygenase. We will also
study the interaction of Sp1 and its interacted protein such as c-Jun, HDAC1 and p300
under TSA treatment in A431 cells.
Role of Sp1 acetylation in the chromatin remodeling
We have known that deacetylation of Sp1 can increase the transcription activity
of 12(S)-lipoxygenase. Next, we want to know its activating mechanism. Because our
preliminary data have shown that HDAC1 and p300 interacted with Sp1 was
increased under PMA treatment in A431 cells, c-Jun may be essential for the
recruitment of HDAC1 to Sp1 to deacetylate the Sp1. Deacetylation of Sp1 may
increase the binding affinity with p300 and recruit to the promoter to acetylate
Histone to induce the chromatin remodeling. To prove this suggestion, plasmids,
pRC-c-Jun-HA and pBSSR-Sp1-His or pBSSR-Sp1 (K703/A)-His will be expressed
with or without PMA treatment in A431 cells. Then, chromatin immunoprecipitation
with anti-Sp1, anti-His, anti-HA, anti-c-Jun, anti-HDAC1, anti-p300 anti-Histone will
be done.
References
1. Huang, W., Zhao, S., Ammanamanchi, S., Brattain, M., Venkasubbarao, K., and
Freeman, J.W. (2005) TSA Induces TGF-beta type II receptor promoter activity
and acetylation of Sp1 by recruitment of PCAF/p300 to a Sp1/NFY complex. JBC
in press.
2. Samson, S.L., and Wong, N.C. (2002) Role of Sp1 in insulin regulation of gene
expression. J Mol Endocrinol. 29, 265-279.
3. Chen, B.K., and Chang, W.C. (2000) Functional interaction between c-Jun and
promoter factor Sp2 in epidermal growth factor-induced gene expression of
human 12(S)-lipoxygenase. Proc Natl Acad Sci USA. 97, 10406-10411.
4. Chang, W.C. (2003) Cell signaling and gene regulation of human 12(S)lipoxygenase expression. Prostaglandins Other Lipid Mediat. 71, 277-285.
5. Wu, Y., Zhang, X., and Zehner, Z.E. (2003) c-Jun and the dominant-negative
mutant, TAM67, induce vimentin gene expression by interacting with the activator
Sp1. Oncogene 22, 8891-8901.
I-2 Molecular mechanism of interaction between vitamin D receptor and Sp1 in
gene regulation (Wen-Chun Hung)
Recent studies show that lipophilic hormones may induce expression of target
genes in which no hormone receptor response elements are found in their promoter
13
regions. These results suggest that nuclear receptors may physically interact with
classic transcription factors to activate gene expression (1, 2). We have previously
demonstrated that vitamin D3 may stimulate the interaction between VDR and
transcription factor Sp1 to activate the expression of a cyclin-dependent inhibitor
p27Kip1 and to suppress proliferation of cancer cells (3). In the second year, we extend
our finding and try to answer the molecular mechanism by which the VDR/Sp1
complex regulates gene expression. Our results suggest that Sp1 functions as an
anchor protein to bring VDR to the Sp1 binding site in the p27Kip1 promoter and VDR
then recruits the co-activators via its activation domain to stimulate p27Kip1 gene
expression. Microarray analysis identifies several potential target genes which
expression may be controlled by the VDR/Sp1 complex. In the third year, we find that
vitamin D3 treatment may induce VDR dephosphorylation. We have identified several
phosphorylation sites in VDR including Ser 9, 51, 203, and 208 and have constructed
the mutant constructs. Our data indicates that the interaction of VDR and Sp1 is
modulated by phosphorylation. In addition, we identify the expression of Skp-2, an
important regulator that mediates p27Kip1 protein degradation and a potential
oncogene, is suppressed by vitamin D3. We clone the human Skp-2 promoter and
demonstrate that vitamin D3 inhibits Skp-2 via Sp1 binding sites. Moreover, our
results indicate that vitamin D3 enhances the formation of VDR/Sp1 complex to
repress Skp-2. In the last year, we will focus on three specific aims. First, we will
continuously clarify the phosphorylation sites that affect VDR and Sp1 interaction.
Second, we will study the effect of acetylation of VDR on its interaction with Sp1
because our data suggest that VDR can be acetylated in vitro. Finally, we will use
proteomic analysis to investigate the protein complexes that interact with VDR/Sp1
complex to repress gene expression.
Experimental Design and Anticipated Results
Role of VDR phosphorylation on the interaction of VDR and Sp1
The vitamin D receptor (VDR) is molecularly dissected into two discrete
domains, the DNA-binding domain (DBD) and the hormone-binding domain (HBD).
The DBD consists of 2 zinc finger motifs which are located within the first 100
residues of the amino terminus. The remaining 300 residues comprise the HBD and a
poorly conserved hinge region. The HBD, in addition to hormone binding, is also
important for protein-protein contact and for transactivation function. Previous studies
have demonstrated that VDR could be phosphorylated by various protein kinases (4).
The first aim of this study is to investigate whether the phosphorylation status of VDR
modulates its interaction with Sp1. Site-directed mutagenesis will be performed to
replace the different phosphorylation sites of VDR and the interaction between these
VDR mutants and Sp1 will be tested in vitro and in cells. These results will help to
14
clarify whether the phosphorylation status of VDR may affect its interaction with Sp1.
Role of VDR acetylation on the interaction of VDR and Sp1
Our recent studies have demonstrated that VDR could be acetylated in vitro.
Whether VDR is indeed acetylated in vivo is unknown. The second aim of this study
is to investigate whether the acetylation status of VDR modulates its interaction with
Sp1. Site-directed mutagenesis will be performed to replace the potential acetylation
sites of VDR and the interaction between these VDR mutants and Sp1 will be tested
in vitro and in cells. These results will clarify whether acetylation of VDR may affect
its interaction with Sp1 and gene transactivation.
Characterization of the protein complexes that interact with the VDR/Sp1
complex to inhibit gene expression
Our previous works have identified a number of potential genes which
expressions may be controlled by the VDR/Sp1 complex. Some of genes are inhibited
by the VDR/Sp1 complex. We have identified that Skp-2 is a target gene for
VDR/Sp1-mediated gene repression. We will synthesize the DNA probes
corresponding to the Sp1 sites in the Skp-2 promoter and DNA affinity precipitation
assay (DAPA) will be performed to pull down the interaction proteins. Protein
identification will be performed by proteomic analysis with the assistance of the
Proteomic Core Lab.
References
1. Inoue, T., Kamiyama, J., and Sakai, T. (1999) Sp1 and NF-Y synergistically
mediated the effect of vitamin D3 in the p27Kip1 gene promoter that lacks vitamin
D3 response elements. J. Biol. Chem. 274, 32309-32317.
2. Safe, S. (2001) Transcriptional activation by 17-beta-estradiol through estrogen
receptor-Sp1 interaction. Vitam. Horm. 62, 231-252.
3. Huang, Y.C., Chen J.Y., and Hung, W.C. (2004) Vitamin D3 receptor/Sp1
complex is required for the induction of p27Kip1 expression by vitamin D3.
Oncogene 23, 4856-4861.
4. Barletta, F., Freedman, L.P., and Christakos, S. (2002) Enhancement of VDRmediated transcription by phosphorylation: correlation with increased interaction
between the VDR and DRIP205, a subunit of the VDR-interacting protein
coactivator complex. Mol. Endocrinol. 16, 301-314.
15
Sub-project (II) Studies of receptor tyrosine kinases signaling through Stat3/Eps8
in human cancer (Principal Investigators: Ih-Jen Su and Tzeng-Horng Leu)
Figure 1
In the fourth year of this subproject, we will remain in the same format as last
year although the endoplasmic reticulum (ER) stress project has been suggested to
move to Subproject III by reviewers after the last site-visit. As shown in Figure 1,
we will use this model to facilitate our interaction and collaboration. In the
intra-subproject 1, Drs. Nan-Haw Chow and Hsiao-Sheng Liu observed Ron can
localize at the nucleus in human bladder cancer. The nuclear Ron is at
dephosphorylated state and interacts with EGFR. MSP stimulation may disrupt this
interaction. Their studies further indicate that the Ron/EGFR complex might interact
with putative Ap1- and/or Sp1-binding sites suggesting that Ron/EGFR might
function as a transcription factor. Furthermore, Ron could phosphorylate Stat3 and
overexpression of Stat3 promotes its biological function. The significance and how
Ron functioning in the nuclei will be focused in this year. In the intra-subproject 2,
Dr. Tzeng-Horng Leu observed Eps8, Src, and FAK are parallel overexpressed in
human colorectal tumors. Furthermore, Eps8 expression is correlated with the
growth rate and FAK expression in SW620 cells. Therefore, he would like to know
whether FAK participates in Eps8-mediated cell growth and metastasis of colorectal
cancer in this year. Furthermore, the relation between Stat3 and Eps8 expression in
colon cancer will be addressed too. In the intra-subproject 3a, Drs. Wu-Chou Su and
Ming-Derg Lai observed that autocrine IL-6 activating Stat3 in lung adenocarcinoma
16
cell line, PC14PE6/AS2 is important for VEGF-induced malignant ascites and pleural
effusion.
Using oligonucleotide microarray analyses, they observed genes
up-regulated and down-regulated in PC14PE6/AS2-S3F (Stat3-negative) cells,
compared with PC14PE6/AS2-Vec(Stat3-positive) cells. To further delineate factors
involved in MPE, they select tissue factor (TF) as the downstream mediator for future
study. In related to the intra-subproject 4, Dr. Lai has observed that ER stress and
expression of HBV pre-S mutant protein induce the COX-2 expression through
regulating pp38MAPK and NF-B transcription factor. In addition, ER stress might
induce altering expression of STAT3 and STAT3, which may affect cellular
behavior. Therefore, he would like to address whether this is a common
phenomenon for many genes and how it occurs. In the intra-subproject 4, Dr. Su
Ih-Jen observed cyclin A induction in HBV preS2 overexpressing cells and in ground
glass hepatocytes (GGHs). Furthermore, the cyclin A is mainly cytoplasmic
localization, which has been implicated in centrosome overduplication and formation
of DNA aneuploidy. The induction of cytoplasmic cyclin A can also occur by ER
stress inducers. They propose the unusual cyclin A expression induced by HBV
pre-S2 mutant proteins may be regulated by ER stress signals and gene transactivation
and he want to clarify this issue.
II-1. The novel mechanisms of tumor stroma and cellular oncogenes in
modulation of bladder carcinogenesis (Nan-Haw Chow and Hsiao-Sheng
Liu)
Background:
The extracellular matrix (ECM) of tissue stroma is known to influence the
proliferation, differentiation, and morphogenesis of normal epithelial cells. The
aberration of ECM contents, as well as its interaction with epithelial cells, may also
impinge upon biological properties of cancer cells. The ECM-derived growth factors
include hepatocyte growth factor (HGF), basic fibroblast growth factor and vascular
endothelial growth factor (1). Recently, we demonstrated the significance of
HGF/c-met pathway in the progression of human bladder cancer (2). This project was
designed to disclose novel mechanism(s) with potential as targets for cancer therapy
in human bladder cancer.
RON receptor tyrosine kinase belongs to c-met receptor family, and is a specific
membrane receptor for macrophage stimulating protein (MSP) (3). Activation of
MSP/RON has been demonstrated to induce epithelial cell migration in vitro (1),
proliferation (4), and tumorigenicity or metastasis in vivo (5). In terms of bladder
cancer, we demonstrated autocrine production of MSP in 6 of 10 uroepihtelial cell
lines, and an elevated MSP in the urine of bladder cancer patients (n = 8) (6).
Moreover, wild-type RON was detected in 5 uroepithelial bladder cancer cell lines,
17
however
UB47 and UB40 two cell line have a 147 bp deletion in the extracellular
domain of RON receptor (RON), which resulted in the aberrant localization of RON
in cytoplasm. In addition, phosphorylation of RON induced by MSP enhances the
migration, proliferation and anti-apoptosis of cancer cells in vitro. Overexpression of
RON was observed in 56 of 165 bladder tumors (33.9%), and level of RON
expression positively correlated with histological grades, muscle-invasion, tumor size
(p < 0.005) and overall patient survival (p < 0.0001). A total of 70 cases (42.4%) also
showed Met overexpression, and the expression status positively associates with
patient survival (p = 0.015). Interestingly, co-expression of RON and Met exhibits
better prognostic significance compared to single or without receptor expression (p =
0.0003) [Chow et al., 2003]. Taken together, activation of MSP/RON pathway, as well
as its interaction with HGF/Met, plays an important role in the progression of human
bladder cancer (6).
Strikingly, confocal microscopy revealed that RON is also located at the nuclei
of cancer cells in an MSP-independent manner, as reported for epidermal growth
factor receptor (EGFR) (7). Moreover, the nuclear RON was at dephosphorylated
status. The result was confirmed by ultracentrifugation of subcellular fractions. As for
mechanism of translocalization, nuclear RON colocalizes with importin 1, importin
1 and dephosphorylated EGFR. But, MSP treatment results in EGFR
phosphorylation accompanied with dissociation of EGFR from RON in the nuclei.
Then siRNA experiments confirmed the significance of EGFR for nuclear localization
of RON. The nuclear RON/EGFR complex was revealed to bind to AP1-binding site
of the promoter using DNA affinity precipitation assay. However, the biological
significance of nuclear RON protein and the importance of EGFR in the RON nuclear
translocalization remain to be determined.
We also demonstrate that RON could phosphorylate Stat3 on tyrosine residue in
the TSGH8301 and J82 inducible-RON cell lines (8). Transient transfection of RON
and Stat3 plasmids showed that Stat3 enhances the MSP/RON-mediated biological
functions, including proliferation, anti-apoptosis, and foci formation. On this base,
Stat3 may involve in the MSP/RON-related signaling event in human bladder cancer.
Whether Stat3 also plays a role in the nuclear translocalization of RON receptor is
under investigation.
Specific Aims:
1. To reveal how and why RON translocate into the nucleus.
2. To clarify the importance of NLS of RON in nuclear translocalization of RON
receptor.
3. To define the biological effect (including proliferation, anti-apoptosis, migration,
18
and invasion) of nuclear RON receptor.
4. To classify whether nuclear RON functions as a transcription factor.
5. To elucidate the significance of EGFR in nuclear translocalization of RON
receptor.
6. To determine the role of Stat3 in the nuclear translocalization of RON receptor.
Experimental Design:
1. We will use immunofluoresent labeling and immunoelectron microscopy to
substantiate the authenticity of nuclear translocalization of RON and EGFR in
TSGH8301 (wild-type RON) and J82 (null RON) cells.
2. Truncation mutants of RON within NLS region will be created by site-directed
mutagenesis to classify the role of RON NLS in nuclear translocation and its
responsive biological functions.
3. The specific pharmaceutical inhibitors and siRNA technique will be utilized to
reveal of 1-integrin, importin and Stat3 in the nuclear import of RON.
4. The potential interacting molecules with nuclear RON will be identified by
GST-fusion protein pull-down assay followed by proteomic profiling.
5. EMSA and CHIP assays will be conducted to clarify the role of nuclear RON as a
transcription factor.
Anticipated Results:
1. We will clarify the mechanisms of ligand-independent nuclear translocalization of
RON receptor.
2. The phosphorylated EGFR may be indispensable for nuclear translocalization of
RON, and Stat3-related signaling may also play a role in nuclear import of RON
receptor.
3. Nuclear RON may function as a transcription factor in mediating the biological
functions via a ligand-independent manner.
References
1. Willett, C. G., Wang, M. H., Emanuel, R. L., Graham, S. A., Smith, D. I., Shridhar,
V., Sugarbaker, D. J., Sunday, M. E. (1998) Macrophage-stimulating protein and
its receptor in non-small-cell lung tumors: induction of receptor tyrosine
phosphorylation and cell migration. Am. J. Resp. Cell Mol. Biol. 18, 489-96.
2. Cheng, H.L., Trink, B., Tzai, T.S., Liu, H.S., Chan, S.H., Ho, C.L., Sidransky, D.,
Chow, N.H. (2002). Overexpression of c-met as a prognostic indicator for
transitional cell carcinoma of the urinary bladder. A comparison with p53 nuclear
accumulation. J Clin Oncol 20, 1544-1550.
3. Wang, M.H., Ronsin, C., Gesnel, M.C., Coupey, L., Skeel, A., Leonard, E. J.,
19
Breathnach, R. (1994). Identification of the RON gene product as the receptor for
the human macrophage stimulating protein. Science 266, 117-119.
4. Gaudino, G., Follenzi, A., Naldini, L., Collesi, C., Santoro, M., Gallo, K.A.,
Godowski, P.J., Comoglio, P.M. (1994). RON is a heterodimeric tyrosine kinase
receptor activated by the HGF homologue MSP. EMBO J 13, 3524-3532.
5. Peace, B.E., Hughes, M.J., Degen, S.J., Waltz, S.E. (2001). Point mutations and
overexpression of Ron induce transformation, tumor formation, and metastasis.
Oncogene 20, 6142-6151.
6. Chow, N.H., Lin, Y.J., Cheng, H.L., Tzai, T.S., Ho, C.L., Chang, T.Y., Dai, Y.C.,
Liu, H.S. (2003) The significance of macrophage stimulating protein (MSP)/RON
signaling pathway in the progression of human bladder cancer. Proceedings of the
American Association for Cancer Research 44, 397.
7. Lin, S.Y., Makino, K., Xia, W., Matin, A., Wen, Y., Kwong, K.Y., Bourguignon, L.,
Hung, M.C. (2001). Nuclear localization of EGF receptor and its potential new role
as a transcription factor. Nat Cell Biol 3, 802-808.
8. Yeh H. H., Liu H. S. and Su W. C. (2005) Ha-ras oncogene induced serine-727
phosphorylation and enhancement of oncogenesity of Stat 3. (Submitted)
II-2. Aberrant expression of Eps8 in human colorectal tumors (Tzeng-Horng
Leu)
Background:
Colorectal cancer is the most common gastrointestinal tumor that comprises a
spectrum of lesions, ranging from benign adenomas to malignant and invasive
carcinomas. Though the genetic events leading to this malignancy has been
elucidated, the underlying molecular mechanisms were still elusive. Our previous
studies have indicated that Eps8, Src, and FAK are parallel overexpressed in human
colorectal tumors. Interestingly, human colorectal cancer cell SW620 exhibits
higher p97Eps8, FAK, and growth rate than SW480 cell. To address the importance of
p97Eps8 in the cell growth of SW620 cells, DNA construct expressing p97 eps8-siRNA
was transfected into SW620 cells and several stable clones, which exhibited reduced
Eps8 expression, were established. And we observed a correlation between p97Eps8
reduction and the decreased ability of cell proliferation of these cell lines both in
culture dishes and in nude mice. Furthermore, accompanying with Eps8 knock
down in these cell lines is the reduction of FAK expression. In addition, Eps8 has
been demonstrated to be one of the TSA-mediated targets in both v-Src transformed
cells (1) and in SW620 cells (unpublished data). In another study, we observed
another HDAC inhibitor, i.e. sodium butyrate, could inhibit the expression of FAK
and Src, and decrease MMP2 and MMP9 secretion in SW620 cells (2). Therefore,
20
the relationship between Eps8 expression and metastatic ability of SW620 cell has a
strong correlation. Since FAK is an important player in integrin-mediated signal
transduction and has been shown to be important for the secretion of MMP2 and
MMP9 in lung adenocarcinoma (3), we would like to know how it participates in
Eps8-mediated cell growth and metastasis of SW620 cells within this year. To
address these issues, the following specific aims will be pursued in this year.
Aim I: To demonstrate the importance of FAK in Eps8-mediated cell growth in
SW620 cells;
Aim II: To demonstrate the importance of Eps8 in the invasion ability of SW620 cells;
Aim III: To illustrate the significance of Stat3 in the regulation of Eps8 expression in
SW620 cells.
Aim I: To demonstrate the importance of FAK in Eps8-mediated cell growth in
SW620 cells.
Since decreased FAK expression occurs in Eps8-knock down SW620 cells by
overexpressing p97eps8-siRNA, we would expect FAK is an important mediator for
Eps8 functioning (i.e. growth control) in SW620 cells. To clarify this issue, we will
introduce FAK expressing construct into these Eps8 knock down cells. Then,
growth rate and several important cell cycle regulators such as p21Waf1/Cip1, p27Kip1,
G1 cyclines (cyclines D and E) within these cells and the vector-transfected control
cells will be compared. The SW620 parental cell will be used as a positive control.
In addition, cell survival contributes a significant part of tumor cell growth. Since
SW620 cell is derived from lymph node-metastasized colorectal cancer, it should
possess ability to avoid anoikis during invasion step. And overexpression of active
FAK in MDCK cells has been shown to be able to overcome anoikis. We expect
induction of FAK expression should contribute this ability in SW620 cell. Therefore,
decreased FAK expression might induce cell death and result in the decreased growth
rate of Eps8-knock down SW620 cells. If so, we will analyze the population of
apoptotic cells in control cells, Eps8-knock down cells and its FAK-overexpressing
cells. Hopefully, increased apoptosis was observed in Eps8 knock down cells and
introducing FAK overexpression in these cell might revert this phenomenon. If there
is any indication that cell death contributes the decreased growth rate of Eps8-knock
down cells, proapototic and antiapototic Bcl2 family proteins will be examined among
these cell lines.
Aim II: To demonstrate the importance of Eps8 in the invasion ability of SW620
cells.
Studies from Funato et. al. indicated that Eps8/IRSp53 complex is important for
21
the regulation of cancer cell motility/invasiveness (4).
In order to address the
involvement of Eps8 in the invasion ability of SW620 cells, invasion assay of the
control cells, Eps8-knock down cells and its FAK-overexpressing cells, as described
above, will be performed. Furthermore, the secretion of MMP2 and MMP9 from
these cells will be analyzed by gelatin zymography.
Aim III: To illustrate the significance of Stat3 in the regulation of Eps8
expression in SW620 cells.
In collaboration with Drs. Su and Lai in the third component of this subproject,
our preliminary data indicates Stat3 may turn on Eps8 expression since dominant
negative Stat3 decrease Eps8 expression in lung cancer PC14PE6/AS2 cell. To
further study Stat3 on the regulation of Eps8 expression in colorectal cancer cells, we
will generate dominant negative (DN) Stat3 expressing SW620 cell lines. If Stat3
activation is required for Eps8 expression, then the Eps8 should be reduced in the
DN-Stat3 expressing cells.
References
1. Leu, T-H, Yeh, H. H., Huang, C.-C., Chuang, Y.-C., Su, S. L., and Maa, M.-C.
(2004) Participation of p97Eps8 in Src-mediated transformation. J. Biol. Chem. 279,
9875-9881.
2. Lee, J. C., Maa, M.-C., Yu, H.-S., Wang, J.-H., Yen, C.-K., Wang, S.-T., Chen, Y.-J.,
and Leu, T.-H. (2005) Butyrate regulates the expression of c-Src and focal adhesion
kinase and inhibits cell invasion of human colon cancer cells. (Submitted)
3. Hauck, C. R., Sieg, D. J., Hsia, D. A., Loftus, J. C., Gaarde, W. A., Monia, B. P.,
and Schlaepfer, D. D. (2001) Inhibition of focal adhesion kinase expression or
activity disrupts epidermal growth factor-stimulated signaling promoting the
migration of invasive human carcinoma cells. Cancer Res. 61, 7079-7090.
4. Funato, Y., Terabayashi, T., Suenaga, N., Seiki, M., Takenawa, T., and Miki, H.
(2004) IRSp53/Eps8 complex is important for positive regulation of Rac and cancer
cell motility/invasiveness. Cancer Res. 64, 5237-5244.
II-3a. Study of the pathogenesis of malignant pleural effusion associated lung
adenocarcinoma in Taiwan and Stat3 as a model gene (Wu-Chou Su and
Ming-Derg Lai)
Background:
In previous studies, we found that autocrine IL-6 activates Stat3 in lung
adenocarcinoma cell lines - PC14PE6/AS2. Overexpression of dominant negative
Stat3 in PC14PE6/AS2 cell reduced its expression of VEGF, induction of microvessel
22
density and vascular permeability, ability for lung metastasis, and formation of
malignant ascites and pleural effusion. Though the reduction of VEGF by
dominant-negative Stat3 may contribute importantly to the phenomenon, there must
be other factors downstream of Stat3, which also contribute to the findings. Using
oligonucleotide microarray analyses, genes up-regulated and down-regulated in
PC14PE6/AS2-S3F (Stat3-negative) cells, compared with PC14PE6/AS2-Vec
(Stat3-positive) cells, were identified. Some of the up-regulated genes were confirmed
by RT-PCR. Among these genes, we will focus on tissue factor (TF) for future study.
We will also explore how the autocrine IL-6 in PC14PE6 cells is regulated.
Specific Aim 1. Role of TF overexpression in lung adenocarcinoma cells with
activated Stat3.
Tissue factor, a transmembrane-receptor protein, is the principal physiological
initiator of blood coagulation. Aberrant TF expression has been detected in a variety
of human tumors, including glioma, breast cancer, non-small cell lung cancer,
leukemia, colon cancer, and pancreatic cancer, but generally is not found in
corresponding normal tissues (1). In addition to coagulation function, experimental
studies have demonstrated that TF also plays an important role in tumor invasion and
metastasis (2,3). There are two Stat3 potential binding site located on 1 Kb upstream
of transcription start site of TF promoter. We therefore expect to know 1) whether
IL-6/JAK/Stat3 pathway regulates TF gene expression, 2) whether Stat3 regulates TF
expression through directly binding to TF promoter, 3) whether the activation of Stat3
is correlated with TF expression in lung adenocarcinoma clinically, and 4) whether TF
in PC14PE6/AS2 cells contributes to the lung metastases, angiogenesis and
generation of MPE by the following approaches:
(1) PC14PE6/AS2 cells will be treated with IL-6 or the JAK inhibitor -- AG490, and
then the expression of TF mRNA and protein and TF reporter gene assay will be
studied.
(2) The Stat3 responding elements on TF promoter will be studied by using reporter
gene assay. Stat3C (active form Stat3) and different TF promoter deletion
constructs will be co-transfected to PC14PE6/AS2 cells or cells without
constitutively activated Stat3 for measurement.
(3) By using Chromatin immunoprecipitation (ChIP) assay, we will examine whether
Stat3 binds to TF promoter in vivo.
(4) Lung cancer tissues, especially samples from patients with lung adenocarcinoma
associated MPE, will be examined by IHC to study the relationship between Stat3
and TF in vivo.
(5) The TF knockdown stable cell lines will be established by transfected with TF
23
siRNA plasmid. In nude mice animal model, the effects of TF on tumor metastasis,
angiogenesis and generation of malignant pleural effusion will be answered.
Specific Aim 2. How autocrine IL-6 in PC14PE6/AS2 cell is regulated?
The human IL-6 promoter contains potential binding sites for a number of
transcription factors, such as AP-1, CAAT enhancer-binding protein, and NF-kB,
those are known to participate in the induction of IL-6 gene expression by various
cytokines (4-6). We’d like to cooperate with investigators in the component project I
to study the regulation of IL-6 in PC14PE6/AS2 cell. We plan to:
(1) study whether Stat3 activation enhance the expression of IL-6 by siRNA assay;
(2) study which transcription factor(s) regulates IL-6 expression in PC14PE6/AS2
cells by the addition of specific inhibitors;
(3) confirm the above findings by transfecting dominant-negative cDNA or siRNA
into the cells.
References
1. Rickles, F. R., Patierno, S., and Fernandez, P. M. (2003) Tissue factor,
thrombin,andcancer. Chest, 124, 58S-68S.
2. Bromberg, M. E., Konigsberg, W. H., Madison, J. F., Pawashe, A., and Garen, A.
(1995) Tissue factor promotes melanoma metastasis by a pathway independent of
blood coagulation. Proc Natl Acad Sci U S A, 92, 8205-8209.
3. Mueller, B. M. and Ruf, W. (1998) Requirement for binding of catalytically active
factor VIIa in tissue factor-dependent experimental metastasis. J Clin Invest, 101,
1372-1378.
4. Asschert JG, Vellenga E, Ruiters MH, de Vries EG. (1999) Regulation of
spontaneous and TNF/IFN-induced IL-6 expression in two human ovariancarcinoma cell lines. Int J Cancer, 82, 244-249.
5. Vanden Berghe W, De Bosscher K, Boone E, Plaisance S, Haegeman G. (1999)
The nuclear factor-kappaB engages CBP/p300 and histone acetyltransferase
activity for transcriptional activation of the interleukin-6 gene promoter. J Biol
Chem, 274, 32091-32098.
6. Franchimont N, Rydziel S, Canalis E. (2000) Transforming growth factor-beta
increases interleukin-6 transcripts in osteoblasts. Bone, 26, 249-253.
II-3b. ER stress and COX-2 induction (Ming-Derg Lai)
Background:
Previous research work demonstrated that endoplasmic reticulum stress and
expression of mutant pre-S HBV large surface protein induced the COX-2 expression
24
through regulating pp38MAPK and NF-B transcription factor (1). Our preliminary
results indicated that endoplasmic reticulum stress induces alteration of expression of
STAT3 and STAT3, which may affect cellular behavior.
following specific aims as our project in the next year.
We propose t the
Specific Aims:
1. The signal pathway from pp38MAPK to transcription factor NF-B was not
completely characterized currently. We will investigate the potential interaction in
signal transduction and nuclear complex formation in COX-2 promoter during
endoplasmic reticulum stress.
2. Endoplasmic reticulum stress may alter cellular transcription and translation
patterns. We will investigate the cellular mechanism of alteration of balance
between STAT3 and STAT3, and study whether altered expression of different
splicing forms of many other proteins occurred during ER stress, i.e. general
alternative splicing patterns during ER stress.
Experimental Approaches:
All of the following experiments will be performed with (1) artificial ER stress
inducer tunicamycin and Brefeldin A (2) expression of mutant pre-S HBV large
surface protein.
1. Examine the serine/threonine phosphorylation state on NF-kB. The AKT
phosphorylation site on p65 will be examined first, though preliminary results
indicated that AKT may not be involved.
2. Nuclear translocation of p38 and the interacting protein complex will be examined
with co-immunoprecipitation during ER stress.
3. The interaction complex on COX-2 promoter during ER stress will be studied
with Chromatin immunoprecipitation.
4. Protein and mRNA expression of STAT3 and STAT3 will be investigated.
Proteosome inhibitor will be employed to determine the role of proteolysis in
conversion between STAT3 and STAT3
5. PCMV-driven expression of STAT3 and STAT3 cDNAin ML-1 cells will be
used to study the potential in studying whether alternative usage of initiation
codon or variation of translation initiation mechanism is activated during ER
stress.
6. Alternative expression of STAT1, STAT5, and several cytokines isoforms during
ER stress will be investigated.
Anticipated Results:
25
1. Identify the regulatory network between p38MAPK, NF-B, and COX-2
promoter.
2. The isoform conversion mechanism of STAT family proteins during ER stress.
Reference
1. Hung, J. H., Su, I. J., Lei, H. Y., Wang, H. C., Lin, W. C., Chang, W. T., Huang, W.,
Chang, W. C., Chang, Y. S., Chen, C. C., and Lai, M. D. (2004) Endoplasmic
reticulum stress stimulates the expression of cyclooxygenase-2 through activation
of NF-kappaB and pp38 mitogen-activated protein kinase. J. Biol. Chem. 279,
46384-46392.
II-4. Regulation of cyclin A expression by HBV pre-S2 mutant protein in the
pathogenesis of hepatocarcinogenesis (Ih-Jen Su)
Background:
Cyclins are important regulators of the cell cycle. Disruption of the G1/S check
point and cyclins/CDKs function may lead to uncontrolled cell growth, resulting in
the development of cancer. Cyclin A associates with CDK2 and CDC2 kinases in the
nucleus and is responsible for the control of S phase progression, DNA synthesis, and
centrosome duplication. By forming complexes with adenovirus E1A protein,
transcription factors DP-1, E2F and Rb, cyclin A has been implicated in cell
transformation. It has been previously noticed that overexpression of cyclin A was
frequently detected in hepatocellular carcinoma (1), and has been correlated with
tumor relapse (2). In our previous studies, cyclin A can be specifically upregulated by
HBV pre-S2 mutant protein at transcriptional level (3). Overexpression of cyclin A
was consistently demonstrated in ground glass hepatocytes (GGHs) expressing HBV
pre-S2 mutant proteins. The expression of cyclin A in GGHs is consistently
cytoplasmic in pre-S2 mutant transgenic mouse liver. Recently, intracellular
redistribution of cyclin A in cytoplasm have been implicated in centrosome
overduplication, increased DNA ploidy, and detachment of kinetochores. In our
laboratory, we have noticed a consistent abnormal cytoplasmic cyclin A expression by
ER stress inducers. These data suggested that the aberrant expression of cyclin A
induced by HBV pre-S2 mutant proteins may be regulated by ER stress signals and
gene transactivation. This project is therefore designed to clarify the potential dual
signal roles of HBV pre-S2 mutant proteins in the activation of cyclin A. The results
will contribute to understanding the role of pre-S mutants in HBV-related
hepatocarcinogenesis. Three major studies will be performed.
Specific Aim I: To define the transactivation role of HBV pre-S2 mutant protein
26
on cyclin A induction via RB/E2F signals.
At G1/S transition of cell cycle, the expression of cyclin A is under the control of
E2F transcription factor, the activation of which requires the hyperphosphorylation of
RB tumor suppressor. In this project, we aim to define whether HBV pre-S2 mutant
protein can induce RB hyperphosphorylation. Disassociation of RB/E2F complex and
activation of E2F transcription factor after RB hyperphosphorylation will be also
evaluated.
Specific Aim II: To detect RB phosphorylation status and cyclin A expression on
hepatocellular carcinoma.
Hyperphosphorylation of RB tumor suppressor was investigated as the
mechanism of RB inactivation in bladder cancer (4). To evaluate whether
hyperphosphorylation of RB is also evident in hepatocellular carcinoma,
immunostaining of both total RB and hyperphosphorylated RB will be performed on
tissue arrays containing neoplastic iver tissues with or without HBV infection.
Meanwhile, the expression level and cellular localization of cyclin A on hepatocellular
carcinoma will also be evaluated.
Specific Aim III: To define the role of ER stress on cytoplasmic localization of
cyclin A.
While the gene induction of cyclin A by HBV pre-S2 mutant protein is ER
stress-independent, our preliminary result reveals that treatment of ER stress inducers
on HuH-7 cells will render its cytoplasmic localization. To further define the potential
mechanism that affects cyclin A localization, various inhibitors will be used to block
this effect. Among several potential candidate signal pathways, we will focus on the
effect of calcium dependent proteases that are activated during ER stress.
Figure 1
Summary of our expected results on HBV pre-S2 mutants induced activation pathways of cyclin A.
27
References
1. Balczon RC (2001) Overexpression of cyclin A in human HeLa cells induces
detachment of kinetochores and spindle pole/centrosome overproduction.
Chromosoma, 110, 381-392.
2. Chao Y, Shih YL, Chiu JH, Chau GY, Lui WY, Yang WK, Lee SD, Huang TS
(1998) Overexpression of cyclin A but not Skp 2 correlates with the tumor relapse
of human hepatocellular carcinoma. Cancer Res, 58, 985-990.
3. Wang HC, Chang WT, Chang WW, Wu HC, Huang W, Lei HY, Lai MD, Fausto N,
Su IJ: Hepatitis B virus pre-S2 mutant upregulates cyclin A expression and induces
nodular proliferation of hepatocytes. Hepatology in press
4. Chatterjee SJ, George B, Goebell PJ, Alavi-Tafreshi M, Shi SR, Fung YK,
Jones PA, Cordon-Cardo C, Datar RH, Cote RJ (2004) Hyperphosphorylation of
pRb: a mechanism for RB tumour suppressor pathway inactivation in bladder
cancer. J Pathol, 203, 762-770.
28
Sub-project (III) Novel signal transduction mechanisms that mediate
anti-apoptotic effects in patho-biology (Principal Investigator: Ming-Jer Tang)
Apoptosis and anti-apoptosis have become important issues for modern
biomedical science. Mechanisms that trigger apoptosis or anti-apoptosis in cell play
very important roles in morphogenesis during development or in patho-physiological
conditions, such as carcinogenesis or regeneration of specific organ. Apoptosis signals
may come from outside of the cell, work on cell membrane receptor, trigger
intracellular death machinery and finally degrade the integrity of cell structure. In this
subproject, we have collaboratively made some progress in studying signaling
mechanisms regarding to interactions of Fas/Fas-L, substratum rigidity-controlled cell
behaviors, such as regulation of integrin activation, FAK phosphorylation, MAPK and
cell migration, and counteractions of lithium to ceramide-induced cell death. Dr. B. C.
Yang demonstrates that Fas cross-linking could directly activate p38 in T cells in a
caspase 8/3-dependent way and p38 MAPK pathway is an auto feed-back switch in
Fas signaling that dampens the death event in T cells. He proposes to define the types
of ECM on tumor cells that activate p38 and ERK to suppress the Fas-mediated
apoptosis in T cells and to study the role of Fas signal feedback route through p38
kinase in autoimmune disorders, such as rheumatoid arthritis (RA) and systemic lupus
erythmatosus (SLE). Collagen gel via the physical property induced down-regulation
of focal adhesion complex proteins in all cells examined, which is mediated by 21
integrin. Dr. M. J. Tang and C. Y. Chou demonstrate that low rigidity of collagen
fibrils suppresses activation of 1 integrin as well as FAK397 phosphorylation. On the
other hand, low substratum rigidity triggers activation of ERK1/2, which results in
augmented cell migration. They will examine the role of membrane rafts in
mechanosensing mechanism triggered by low rigidity as well as the molecular
mechanism of low rigidity-induced shift from FAK397 activation to Erk1/2 signaling
pathways. Finally, Dr. Y. S. Lin works on the novel mechanism by which lithium
serves to counteract ceramide-induced apoptosis in immune cells and shows that
lithium confers protection from ceramide-induced apoptosis via activation of
MEK/ERK/Hsp70 and inhibition of mitochondrial activation. Lithium promotes cell
survival by inhibiting PP2A activity and caspase-2 activation. Furthermore, GSK-3
is required in ceramide-induced mitochondrial apoptosis. She proposes to explore the
regulatory effect of lithium on kinase and phosphatase functions and to dissect the
mechanisms of the inhibitory effect of lithium on caspase-2-mediated mitochondrial
damage. Results of our studies should have impact in cancer biology, immunology,
and developmental biology.
29
III-1. Suppression of the Fas-mediated death signal in T cells upon contact with
tumors (Bei-Chang Yang)
Background:
Fas/Fas-L system is one of the major apoptosis-mediating signals and plays an
important role in various immune functions. Previously, we have shown a
tissue-dependant effect of Fas-L on tumorigenecity (1, 2). Accumulating data reveal
that extracellular matrix (ECM), constituting mainly the tissue/tumor
microenvironments, can directly initiate a variety of signals. For instance, integrin
binding activates the MAPK/ERK pathway in T lymphocytes (3). Integrin can
modulate transmission of signals downstream of growth factor receptors by
internalization of membrane domains (4). Recently, we observed that direct cell
contact with tumor of various types reduced the Fas signal-mediated apoptosis in T
cells, both direct Fas crosslinking with agonistic antibody and activation-induced cell
death. Decreases in the characteristics of apoptosis including caspase-8, -9, and -3
activation, loss of plasma asymmetry, cell shrinkage, and loss of mitochondria
membrane potential in Jurkat cells were accompanied by increases in phosphorylation
of ERK and p38 upon coculture with glioma cells. Simultaneously inhibit the kinase
activities of ERK, PI3K, and p38, respectively, could abrogate the inhibited-apoptosis
phenotype in Jurkat cells (see figure, manuscript in preparation). These findings
suggest that ECM could also modify immune surveillance by affecting viability of
immune cells. In the first part of this project, we specifically investigate how and
what tumor/ECMs shape Fas signaling.
Summary of our study on the inhibition
Second part of this project is on novel of Fas-mediated apoptosis in T cells
feedback regulation of Fas signaling. Some
Fas-expressing cells do not undergo cell death but
proliferation and differentiation upon Fas
stimulation. In agreement with those findings, we
have found that Fas signaling stimulated IL-10
expression in T cells through a PKA-independent
pathway (5). In addition, we found that Fas
cross-linking could directly activate p38 in T cells
in a caspase 8/3-dependent way which might form
an auto-feedback route to shut down apoptosis cascade. Comparing to the
Fas-mediated apoptotic pathway through caspase activation, how Fas signal activates
kinase activities and what is its biological significance are still poorly understood.
Because altered p38 kinase activity has been correlated with several autoimmune
diseases, we speculate that the feedback route of Fas signaling plays a role in
autoimmunity.
30
Specific aims of this year:
(1) define the types of ECM on tumor cells that activate p38 and ERK to
suppress the Fas-mediated apoptosis in T cells; (2) study the role of Fas signal
feedback route through p38 kinase in autoimmune disorders, such as rheumatoid
arthritis (RA) and systemic lupus erythmatosus (SLE).
Experimental Design and Anticipated Results:
1. How and what tumor ECMs activate the p38 and ERK to suppress Fas-mediated
apoptosis in T cells?
A. tumor ECM and its ligands on lymphocytes
To identify the responsible ECM for the protective signaling, a panel of blocking
antibodies specific for particular ECM/integrins will be used to interrupt the ECM/
integrins engagement between Jurkat cells and tumor cells. The study about
ECM/integrins is collaborated with Drs. MJ Tang and WJ Chuang. Moreover,
complimentary study using 2-dimensional gel/mass spectrometry/sequence data bass
searching algorithms (Core facility 1: Mass spectrometry facility) has identified some
interesting proteins in Jurkat cells upon contact with tumor cells. These approaches
allow us to answer questions about how many genes are induced by ECM and the
putative ECM/integrins signaling pathway
B. the mechanism of p38 and ERK to inactivate caspases
The Fas signal-activated caspase-8, -9 and -3 in Jurkat cells were reduced upon
coculture with tumor cells along with enhanced phosphorylation of p38 and ERK.
Immunoprecipitation pull-down assay will be performed to study whether
phosphorylation/de-phosphorylation process is involved in regulating caspase
activities. The phosphorylation status of caspase-8, -9 and -3 will be determined by
phosphor-serine/threonine antibodies.
C. characterization of tumor infiltrating lymphocytes (TILs) in vivo
Putative Fas-signal, survival-associated genes in TILs in human specimen will
be determined. Our preliminary data obtained by intracellular stain/FACS analysis
indicated that some infiltrated T cells isolated from fresh human gastric carcinoma
have altered caspase 3 activity. Taking the advantage of tumor tissue bank established
in our medical center and the capacity of tumor center in the NCKU hospital, we have
opportunity to systemically analyze the apoptosis, p38, ERK, and caspase 3 activates
of TILs by immunohistochemical stain method in different tumor samples of different
types and differently tumor stages. We will answer the question whether activations
of p38, ERK, and caspase 3 are associated with apoptosis in those TILs.
31
(2) What is the role of Fas signal feed-back route through p38 kinase in autoimmune
disorders?
A. Fas signaling to activate p38 kinase
Our preliminary data showed that caspase 8, but not caspase 2 activity was
required for the phosphorylation of p38 in Fas signaling. We will knock down the
expressions of caspase 8, caspase 3 and FADD genes by siRNA strategy.
Immunoprecipitation pull-down assay for Fas DISC will be performed to verify if p38
is physically associated with Fas death complex. If this speculation was turned out to
be positive, colocalization of p38 and caspases/FADD in Jurkat T cells will be
visualized by confocal microscopy.
B. Evaluation of Fas signaling in T cells of RA and SLE patients
We observed that p38 was dephosphorylated during T cell activation. Activation
induced cell death, which is partly Fas-mediated, could be enhanced by inhibitor for
p38. Moreover, we found an elevated phosphor-p38 in T cells isolated from
peripheral blood and synovial fluid of RA patients. We will analyze the Fas signaling
and activation cell death of T cells of RA and SLE patients. Correlation of defect in
the p38-associated auto-feedback route of Fas signal with a prolonged survival of
activated T cells of RA and SLE patients will be established. Results obtained will
shed light on the etiology of autoimmunity.
References
1. Chen YL, Wang JY, Chen SH, and Yang BC (2002) Granulocytes mediates the
Fas-L-associated apoptosis during lung metastasis of melanoma that determines the
metastatic behavior. Br. J. Cancer 87, 359-365.
2. Chen YL, Chen SH, Wang JY, and Yang BC (2003) FasL on tumor cells mediates
inactivation of neutrophils. J Immunol 171, 1183-1192.
3. Gendron S, Couture J, and Aoudjit F (2003) Integrin alpha2/beta1 inhibits
Fas-mediated apoptosis in T lymphocytes by protein phosphatease 2A-dependent
activation of the MAPK/ERK pathway. J Biol Chem 278, 48633-48643.
4. del Pozo MA, Alderson NB, Kiosses WB, Chiang HH, Anderson RGW, and
Schwartz MA (2004) Integrins Regulate Rac Targeting by Internalization of
Membrane Domains. Science 303, 839-842.
5. Yang BC, Hor WS, Lin HK, Hwang JY, Lin YP, Liu MY, and Wang YJ (2003)
Stimulation of IL-10 expression in T cell lines upon contact with human glioma
cells, that is mediated by Fas signaling through a PKA- independent pathway. J
Immunol 171, 3947-3954.
III-2: Signaling mechanisms of low rigidity-induced inhibition of FAK397
32
phosphorylation and activation of Erk1/2 (Ming-Jer Tang and
Cheng-yang Chou)
Background:
Physical environment has been considered as an important factor in regulating
cellular behavior. How mechanical impacts affect cell behaviors is a less studied issue.
We have shown that collagen gel, as a soft substratum, down-regulates the level of
focal adhesion complex proteins through 21 integrin (1). Her we continue to
delineate how a cell responses to low rigidity, particularly to examine whether the
subrstratum rigidity of collagen gel affect cell migration and the signal transduction
mechanisms involved in low rigidity induced cell responses. When MDCK cells were
cultured on collagen gel, phosphorylation sites (407, 577, 861, and 925) of FAK were
activated, only FAK397 phosphorylation levels remained low or unaltered. Low
rigidity-induced decrease in FAK397 phosphorylation could be observed in various cell
lines including fibroblasts and transformed cells. In addition, 1 integrin activation
was down-regulated under low substratum rigidity. Disruption of actin cytoskeleton
by cytochalasin D blocked FAK397 phosphorylation but not 1 integrin activation,
while disruption of microtubules by colcemide had no effect. MCD, a lipid raft
inhibitor, inhibited 1 integrin activation in cells cultured on rigid substratum.
However, the level of FAK397 phosphorylation remained unaffected. Taken together,
our data provides a new concept that FAK397 phosphorylation needs internal force
provided from actin filaments, but 1 integrin activation requires preferentially
external force from rigid substratum and lipid raft may regulate this process (2). We
further showed that low rigidity of collagen gel triggered cell migration in all cell
lines examined. Low rigidity-induced cell migration was mediated by a delayed onset
of Erk-1/2 phosphorylation localized on focal adhesions (3). Collagen gel induced
phosphorylation of ERK1/2 within 1 hour and the induction could last for more than 8
hour. Inhibition of collagen-induced ERK1/2 phosphorylation by MEK inhibitor,
UO126, resulted in round up morphology. In view of these results, we will examine
the role of membrane rafts in mechanosensing mechanism triggered by low rigidity as
well as the molecular mechanism of low rigidity-induced shift from FAK397
activation to Erk1/2 signaling pathways.
(1) The role of membrane rafts in mechanosensing mechanism triggered by low
rigidity
We will investigate the mechano-sensing mechanisms whereby cells utilize to
detect the rigidity of collagen fibrils. MDCK cells will be cultured under different
conditions (collagen gel, collagen gel-coated dish and dish). Whether the membrane
microdomain provides signals leading to 1 integrin activation will be analyzed by
33
fractionation with the membrane lysate being analyzed by sucrose gradient under
ultracentrifuge. Membrane protein markers, like caveolin-1 and Na-K,ATPase will be
used as indicator for rafts and non-rafts, respectively. MCD, a lipid raft inhibitor,
will be used to investigate whether it affects the level of 1 integrin activation, FAK
phosphorylation and activation of MAPK signaling pathways. To elucidate whether
the internal force provided from actin filaments and microtubules affected 1 integrin
activation and FAK397 phosphorylation, we will employ cytochalasin D and colcemide.
To examine whether low rigidity-induced down-regulation of 1 integrin activation or
FAK397 phosphorlyation is mediated through FAK or DDR1, wild type and dominant
negative FAK or DDR1 stably transfected MDCK cells will be employed (5).
(2) Molecular mechanism of low rigidity-induced shift from FAK397 activation
to Erk1/2 signaling pathways
The collagen gel-induced ERK1/2 phosphorylation is present in focal adhesion in
addition to the nuclei of the cell, indicating that cytosolic ERK1/2 activation may lead
to cell spreading. Moreover, filipin III, a specific inhibitor for caveolae, completely
alleviates the collagen gel-induced ERK1/2 phosphorylation. Taken together, low
rigidity of collagen fiber induces activation of ERK1/2 may be the result from
inactivation of FAK397 phosphorylation. In order to test this hypothesis, we will
employ the FAK397 mutant as well as FAK397 constitutive active form to dissect the
cause-effect relationship between suppression of FAK397 phosphorylation and Erk1/2
activation triggered by low rigidity.
(3) The role of TFIID in low rigidity-induced decrease in protein synthesis rates
We have found that low rigidity of collagen gel suppresses protein synthesis rates
in all cells examined. In addition, we have also demonstrated that low-rigidity results
in down-regulation of TFIID mRNA and protein levels. It is likely that the low
rigidity-induced decrease in protein synthesis rate could be mediated by
down-regulation of TFIID (4).
References
1. Y. K. Wang, Y. H. Wang, C. Z. Wang, J. M. Sung, W. T. Chiu, S. H. Lin, Y. H.
Chang and M. J. Tang (2003) Rigidity of collagen fibrils controls collagen
gel-induced down-regulation of focal adhesion complex proteins mediated by
21 integrin. J. Biol. Chem. 278, 21886-21892.
2. W. C. Wei, Y. K. Wang and M. J. Tang. Substratum rigidity controls 1 integrin
activation and FAK phosphorylation. (manuscript in preparation)
3. Y. C. Hsu, W. T. Chiu, Y. K. Wang and M. J. Tang. Mechanical property of
34
collagen fiber-induced cell spreading and migration are mediated by
phosphorylation of ERK1/2. (manuscript in preparation)
4. Y. K. Wang and M. J. Tang. Low rigidity of collagen fibrils down-regulates TFIID
and decreases protein synthesis rate. (manuscript in preparation)
5. C. Z. Wang, H. W. Su, Y. C. Hsu, M. R. Shen and M. J. Tang (2005) SHP-2
mediates DDR1-induced suppression of STAT3 tyrosine phosphorylation, cell
migration and branching tubulogenesis. EMBO J (in revision)
III-3: Molecular mechanism of ceramide-induced apoptosis: the anti-apoptotic
role of lithium (Yee-Shin Lin)
The goal of this research is to study the anti-apoptotic roles of lithium against
ceramide-induced apoptotic signaling pathways. Apoptotic signaling mediated by
ceramide offers new insights into mechanism of action of chemotherapy and
radiotherapy in antitumor activity [1, 2]. Our previous results showed sequential
activation of caspase-2 and -8 before mitochondrial damage during ceramide- and
etoposide-induced apoptosis [3]. Bcl-2 knockdown by siRNA or inhibition by
HA14-1 resulted in an autonomic activation of caspase-2, providing direct evidence of
a negative regulatory role of Bcl-2 on caspase-2. In addition, Bcl-2 dephosphorylation
mediated by ceramide-activated protein phosphatase 2A (PP2A) caused caspase-2
activation and mitochondrial apoptosis [4]. Lithium, a drug used for neuroprotection,
was shown to confer protection against ceramide-induced T cell apoptosis by
activating survival kinases such as MEK/ERK [5] and by inhibiting PP2A activity and
caspase-2 activation [6]. Further studies indicated that the inhibition of GSK-3 by
lithium may also be involved in the cell protection against apoptotic effects of
ceramide [7].
Thus, the anti-apoptotic effects of lithium involve both an enhancement of cell
survival pathway and an inhibition in cell death pathway. The specific aims of the
fourth year proposal are: (1) to explore the regulatory effect of lithium on kinase and
phosphatase functions, and (2) to dissect the mechanisms of the inhibitory effect of
lithium on caspase-2-mediated mitochondrial damage.
Effects of lithium on kinase and phosphatase functions
Our previous results showed that ceramide caused cell apoptosis by inducing a
turn-off mechanism of survival kinases such as MEK, ERK, and Akt [5] and a turn-on
mechanism of proapoptotic pathways through activating PP2A [6] and GSK-3 [7].
Previous report indicated that ceramide induced PP2A activation associated with
mitochondrial damage and cell death [8]. Cell apoptosis was inhibited by the
pretreatment of okadaic acid, suggesting the involvement of PP2A activation in
35
ceramide-induced apoptotic pathway. Indeed, PP2A may play a role in the
downregulation of survival signal via MEK, ERK, and Akt dephosphorylation.
Furthermore, our study showed that the activation of PP2A was required for
ceramide-induced GSK-3 activation. To dissect the anti-apoptotic role of lithium,
lithium-conferred inhibition on PP2A activation was demonstrated. The possible
mechanisms of lithium in the inhibition of PP2A activation followed by blockage of
ceramide-induced apoptosis will be further explored.
Lithium, an inhibitor on GSK-3, causes GSK-3 inactivation directly by
2+
Mg -ATP competition and indirectly through an enhancing phosphorylation effect on
GSK-3 serine 9. Our studies indicate that GSK-3 activation is essential for
ceramide-induced apoptosis. Although PP2A inactivation may suppress
ceramide-mediated GSK-3 activation and apoptosis, the underlying mechanisms
mediated by lithium remain undefined. Our preliminary data show that
lithium-induced p38 MAPK activation may be required for GSK-3 phosphorylation.
Using specific inhibitors and siRNA technique, the novel mechanisms of GSK-3
inactivation by lithium-induced p38 MAPK activation are to be investigated.
Moreover, the mechanisms of lithium-mediated p38 MAPK activation and its effects
on providing cell survival against ceramide remain further investigation.
Effects of lithium on caspase-2-mediated mitochondrial damage
Recently, caspase-2 was shown to act upstream of mitochondria in stress-induced
apoptosis. Activation of caspase-8, a key event in death receptor-mediated apoptosis,
also has been demonstrated in death receptor-independent apoptosis. The regulation of
these initiator caspases, which trigger the mitochondrial apoptotic pathway, is unclear.
In the study of the molecular mechanisms of apoptosis induced by ceramide, we
found that caspase-2 acted upstream of caspase-8 before mitochondrial damage [3].
The mechanisms of initiator caspases activation following ceramide treatment remain
investigation. How caspase-2 regulates caspase-8 activation is also unknown.
Interestingly, lithium inhibited the activation of initiator caspase-2 and -8. The
mechanism of anti-apoptotic effect of lithium by inhibiting caspase-2 activation is
also unclear. Our study showed that lithium confers cell survival against ceramide by
inhibiting PP2A, GSK-3, and caspase activation and by activating survival factors
including MEK/ERK. These results suggest a multiple regulatory pathways of
ceramide on caspase-2 activation that leads to mitochondrial damage.
First, to explore the regulation of PP2A on caspase-2 activation, the relationship
between phosphatase activity and initiator caspases activation will be examined.
Using specific phosphatase inhibitors and antisense or RNAi techniques, the roles of
PP2A and other phosphatases on initiator caspases activation in ceramide-induced
36
apoptotic signaling pathways will be dissected. Second, the regulatory effects of
PP2A on Bcl-2 and of Bcl-2 on caspase-2 will be investigated. Our preliminary results
show that Bcl-2 and caspase-2 can be co-precipitated. Whether Bcl-2 and caspase-2
can directly interact with each other or whether adaptor proteins are necessary for
Bcl-2 and caspase-2 binding requires further studies. Third, the requirement of
GSK-3 for ceramide-induced caspase-2 activation was demonstrated. The
mechanism of action of GSK-3 on caspase-2-mediated mitochondrial apoptosis will
be further investigated.
References
1. Jaffrezou, J. P., and G. Laurent. 2004. Ceramide: a new target on anticancer
research? Bull. Cancer 91, E133-161.
2. Ogretmen, B., and Y. A. Hannun. 2004. Biologically active sphingolipids in cancer
pathogenesis and treatment. Nat. Rev. Cancer 4, 604-616.
3. Lin, C. F., C. L. Chen, W. T. Chang, M. S. Jan, L. J. Hsu, R. H. Wu, M. J. Tang, W.
C. Chang, and Y. S. Lin. 2004. Sequential caspase-2 and caspase-8 activation
upstream of mitochondria during ceramide- and etoposide-induced apoptosis. J.
Biol. Chem. 279, 40755-40761.
4. Lin, C. F., C. L. Chen, W. T. Chang, M. S. Jan, L. J. Hsu, R. H. Wu, Y. T. Fang, M.
J. Tang, W. C. Chang, and Y. S. Lin. Bcl-2 rescues ceramide- and
etoposide-induced mitochondrial apoptosis through blockage on caspase-2
activation. (submitted)
5. Jan, M. S., L. J. Hsu, C. F. Lin, C. L. Chen, and Y. S. Lin. Lithium confers
protection from ceramide-induced apoptosis via activation of MEK/ERK.
(manuscript in preparation)
6. Chen, C. L., C. F. Lin, C. W. Chiang, M. S. Jan, M. J. Tang, W. C. Chang, and Y. S.
Lin. Lithium blocks ceramide-induced mitochondrial damage by inhibiting
caspase-2 activation and protein phosphatase 2A methylation. (manuscript in
preparation)
7. Lin, C. F., C. L. Chen, C. W. Chiang, M. S. Jan, M. J. Tang, W. C. Chang, and Y. S.
Lin. Glycogen synthase kinase-3beta modulates ceramide-induced T cell
mitochondrial apoptosis. (manuscript in preparation)
8. Ruvolo, P. P., X. Deng, T. Ito, B. K. Car, and W. S. May. 1999. Ceramide induces
Bcl2 dephosphorylation via a mechanism involving mitochondrial PP2A. J. Biol.
Chem. 274, 20296-20300.
37