Download Functional role of c-Jun/PP2B in regulation of gene expression

Post-translational modification of Sp1 in gene regulation
Jan-Jong Hung, Hui-Ling Tsai, and Wen-Chang Chang
Department of Pharmacology, College of Medicine, National Cheng Kung University
Sp1 is a basic transcriptional factor, which binds to the GC-rich region in the
promoter of target gene. It is involved in transcription of numerous genes by
recruiting other transcriptional factors to the promoters of target genes. Recently,
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. 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 showed that transcription of
12(S)-lipoxygenase is regulated through interaction of c-Jun and Sp1 upon PMA or
EGF treatment in A431 cells. Recently, we also found that Sp1 can recruit Hsp90αto
the promoter of the 12(S)-lipoxygenase to modulate its transcriptional activity (Hung,
et al., 2005). In addition, we also found that the deacetylation of Sp1 is important for
the induction of 12(S)-lipoxygenase under PMA treatment and characterized the
mechanism (Hung, et al., 2006). The previous studies indicate that Tricostatin A (TSA)
is a stimulator of p21WAF1/CIP1. Recently, in our study, we found that HDACs, class I,
have the positive effect in expression of p21 WAF1/CIP1 in p53-dependent and
-independent cells. The experiments of HDACs overexpression and that of HDACs
knockdown in p53-dependent and –independent cells revealed that HDACs were
positive effect in the expression of p21 WAF1/CIP1. Furthermore, we studied the
mechanism and found that the recruitment of p300 to Sp1 binding region of the p21
WAF1/CIP1
promoter was increased obviously. At last, overexpression of Sp1(K703/A)
which can not be acetylated could increase the transcriptional activity of p21 WAF1/CIP1.
Taken together, HDACs, class I, may take a positive effect in the regulation of p21
WAF1/CIP1
through Sp1-dependent pathway.
However, several points in our studies are still unclear and need study further.
First, it is still unclear how many genes in cells were regulated through this
mechanism suggested in our study. Second, how to affect the transcriptional activity
of 12(S)-lipoxygenase by the Hsp90αthrough the Sp1. Third, there is a high
possibility predicted by computer that Sp1 also can be sumoylation, and our
preliminary result also revealed that Sp1 can be sumoylated in vitro. Therefore,
identification of the sumoylated site(s) in vivo and its role in the gene regulation need
to study in the future. Fourth, the regulation between the modification of Sp1 such as
phosphorylation, acetylation and sumoylation in cells is still unknown.
Functional role of c-Jun/PP2B in regulation of gene expression
Ben-Kuen Chen, Chi-Chen Huang, and Wen-Chang Chang
Department of Pharmacology, College of Medicine, National Cheng Kung University
c-Jun/Sp1 interaction is essential for growth factor- and phorbol 12-myristate
13-acetate (PMA)-induced genes expression, including human 12(S)-lipoxygenase,
keratin 16, cytosolic phospholipase A2, p21WAF1/CIP1 and neuronal nicotinic
acetylcholine receptor 4. We found that the mechanism used to regulate the
c-Jun/Sp1 interaction induced by PMA and EGF is mediated by calcineurin (PP2B).
Inhibition of calcineurin (PP2B) by using cyclosporin A and PP2B small interfering
RNA resulted in attenuating the PMA- and EGF-induced gene expression and
c-Jun/Sp1 interaction. PP2B bound and dephosphorylated the phospho-TAM-67 and
interacted with c-Jun in PMA-treated cells. To investigate domains involved in the
regulation of c-Jun and PP2B interaction, immunoprecipitation and FRET assays were
used. Both calmodulin and calcineurin B domains of PP2B were involved in the
binding with c-Jun. Since PMA also induced the interaction between c-Jun and PP2B,
we want to analyze the function and mechanism involved in the regulation of
c-Jun/PP2B complex. PP2B was sumoylated both in PMA-treated cells and in an in
vitro enzymatic assay. These results indicated that PMA induced the interaction
between c-Jun and PP2B may be through the posttranslational protein modification. In
addition, PMA-induced PP2B sumoylation also indicated the possibility of novel
function of PP2B in the gene expression.
Regulation of gene expression by the VDR/Sp1 complex
Yu-Chun Huang, Jing-Yi Chen, Hsuen-Tsen Cheng, Nan-Guai Tsai, Hui-Chiu Chang,
and Wen-Chun Hung
Graduate Institute of Medicine, Kaohsiung Medical University, and Institute of
Biomedical Sciences, National Sun Yat-Sen University
Recent studies show that lipophilic hormones may induce expression of target
genes in which no hormone receptor response elements are found in their promoter
regions. These results suggest that nuclear receptors may physically interact with
classic transcription factors to modulate gene expression. In the past four years, we
address the functional interaction of vitamin D3 receptor (VDR) and Sp1 in the
regulation of gene transcription. The main findings of our study include: (1) VDR
may bind indirectly to DNA via interaction with Sp1 transcription factor. In addition,
the VDR/Sp1 complex may activate or repress gene transcription via the Sp1 site in
the promoter. A number of target genes such as p27 and Skp-2 which expressions are
controlled by the VDR/Sp1 complex have been identified. (2) The functional role of
VDR and Sp1 in the complex has also been characterized. Our results suggest that
Sp1 functions as an anchor protein to bring VDR to the Sp1 binding site in the gene
promoter and VDR then recruits the co-activators or co-repressor via its activation
domain to stimulate or repress gene transcription. (3) Post-translational modification
of VDR may modify the transcriptional activity of this receptor. We find that VDR
can be sumoylated and acetylated in vitro and in vivo. Sumoylation of VDR enhances
VDR/RXR-mediated transcriptional activation as determined by promoter activity
assays. Taken together, our study strongly suggests that VDR may bind with Sp1 to
form the VDR/Sp1 complex and modulate gene expression via the Sp1 site in the
promoter.
Post-transcriptional control of thrombomodulin expression: RNA-binding
protein HuR and poly(C)-binding protein 1 bind with the 3’-untranslated region
and enhance the messenger RNA stability
Joseph T. Tseng, and Wen-Chang Chang
Department of Pharmacology, College of Medicine, National Cheng Kung University
Thrombomodulin (TM), recognized as an important anticoagulant factor, is also
expressed by a wide range of tumor cells. Overexpression of wild-type TM decreases
cell proliferation in vitro and tumor growth in vivo. The cDNA sequence of TM shows
a very long 3’-untranslated region (around 2 Kbps). However, the functional role of
this region involved in the regulation of TM expression is not cleared. Here we
showed that the post-transcriptional control is an universal mechanism in regulating
the TM mRNA expression in HUVEC and A431 cell lines. Under different stimulator
treatments, the TM mRNA stability will be enhanced around 2-fold. From the
RNA-EMSA experiment, we identified a UC-rich region in the 3’-UTR of
thrombomodulin mRNA binding with the protein complex. It indicated this region
involved in the regulation of mRNA stability of TM. Furthermore, two major binding
proteins HuR, poly(C)-binding protein 1 (PCBP1), were identified by in vitro binding
assay. RNA-immunoprecipitation assay indicated that HuR interacted with TM
mRNA in vivo and that this interaction increased following stimulator treatments.
Suppression of HuR and PCBP1 expression by RNA-interference blocked the
accumulation of TM mRNA. These results demonstrated that HuR and PCBP1
coordinated the regulation of TM mRNA stability. Regarding the signal pathway for
regulating the TM mRNA stability, Ly294002, a PI3-Kinase inhibitor, was shown to
block the stabilization mechanism and enhance the degradation of TM mRNA.
Therefore, PI3-Kinase played an important role in mediating the EGF signal induced
mRNA stability.
Molecular mechanism of EGF-enhanced Aurora-A gene expression in tumor cells
Liang-Yi Hung, Minli Wu, Chien-Hsien Lai, and Wen-Chang Chang
Department of Pharmacology, College of Medicine, National Cheng Kung University
Human Aurora-A kinase is highly expressed in a lot of malignant tumor cells and
cancer cell lines. According the documents previously reported, the human Aurora-A
gene is a potential oncogene. The increased level of human Aurora-A kinase in
malignant tumors and cancer cell lines is through the DNA amplification, protein
stabilization and transcriptional up-regulation. The detailed mechanism of
transcriptional regulation of human Aurora-A gene in tumors is still unclear. In this
study, when treated cultured cell lines with EGF, only in EGFR (EGF receptor)
overexpressed cell lines (A431, MDA-MB-231, MDA-MB-468 and LS174T), the
Aurora-A kinase is up-regulated. Analyze the promoter sequence of Aurora-A gene,
there are five predicted ATRSs (the AT-rich sequence, which is the predicted
EGFR-recognition sequence) are located upsatream of the promoter region, and three
of them contain the predicted STATs (signal transducers and activators of
transcription)-binding sequence. The immunoblotting analysis demonstrated the
nuclear location of EGFR in these EGFR highly expressed cultured cell lines, except
LS174T. Using chromatin immunoprecipaitation (ChIP) assay, we observed
significant EGF-induced recruitment of nuclear EGFR to the Aurora-A promoter in
A431 cells. DNA-affinity purification assay (DAPA) and ChIP assay further showed
the binding of Sp1 in the predicted Sp1-binding site of Aurora-A promoter.
Surprisingly, c-Jun is also recruited to the promoter region of Aurora-A which lacks
the conserved AP1 binding site. The regulatory mechanism about how EGF-enhanced
Aurora-A kinase expression in a lot of malignant tumor cells is under investigated.
The significance of macrophage stimulating protein/RON in human bladder
carcinogenesis
Pei-Yin Hsu1, Nan-Haw Chow2, and Hsiao-Sheng Liu3
1
Institute of Basic Medical Sciences, 2 Department of Pathology, 3 Department of
Microbiology and Immunology, College of Medicine, Nation Cheng Kung University
The extracellular matrix (ECM) of tissue stroma affects the proliferation,
differentiation, and morphogenesis of normal epithelial cells. Moreover, aberration of
ECM components, as well as its interaction with epithelial cells, also impinges upon
the biological properties of cancer cells. A number of growth factors are deposited in
the ECM, including hepatocyte growth factor (HGF), basic fibroblast growth factor
(bFGF) and vascular endothelial growth factor (VEGF). Our previous study revealed
the significance of HGF/c-Met signaling pathway in the progression of human bladder
cancer. This project thus was designed to explore novel mechanism(s) involved in
bladder carcinogenesis as well the potential for cancer therapy.
RON, one of the members of c-Met receptor family, is a distinct receptor
tyrosine kinase. Previous studies showed that overexpression of RON positively
relates to progression of various carcinomas. Our cohort study also revealed that
RON-associated signaling involves in cancer progression, and that co-expression of
RON and Met plays more important role in bladder carcinogenesis. RON
phosphorylation induced by MSP enhances the migration, proliferation and
anti-apoptosis of bladder cancer cells. Strikingly, de-phosphorylated forms of both
wild-type and truncated RON were detected in the nuclei of cancer cells. We thus
propose that nuclear translocalization of RON receptor may play an important role in
the progression of bladder cancer. In addition, translocalization of de-phosphorylated
RON were found to be mediated by ligand-independent mechanism. The nuclear
RON was co-localized with importin 1, importin 1 and de-phosphorylated EGFR.
Nevertheless, MSP treatment results in EGFR phosphorylation together with
dissociation from nuclear RON, implying a cross-talk between RON and EGFR. The
siRNA experiments confirmed the importance of EGFR on nuclear translocalization
of RON. We also found that collaboration of RON and EGFR contributes to
RON-mediated biological effects in human bladder cancer cells.
To clarify the significance of nuclear localization signal (NLS) in the nuclear
translocalization of RON, site-directed mutagenesis assay was performed on both Nand C-terminal NLSs of RON. Our preliminary results showed that R306Q at
N-terminal and both R1388T and R1389T at C-terminal NLS of RON effectively
suppress the translocalization of RON to the nuclei, supporting a classical
NLS-dependent mechanism. What is more, both proliferation and anti-apoptosis of
NLS mutants were apparently repressed compared with wild-typed RON transfectants,
suggesting that nuclear RON might play a crucial role in the proliferation and
anti-apoptosis of cancer cells.
Aberrant expression of Eps8 in human colorectal tumors
Tzeng-Horng Leu
Department of Pharmacology, College of Medicine, National Cheng Kung University
Our early studies have indicated that: (i) Eps8 (i.e. both p97Eps8 and p68Eps8)
overexpression occurs in v-Src-transformed cells; (ii) ectopically expressed p97Eps8
promotes cell transformation of C3H10T1/2 fibroblast; and (iii) ablation of Eps8
expression in v-Src-transformed cell, IV5, reverts its growth advantage character.
Thus, p97Eps8 may play an important role in Src-mediated transformation. Indeed,
when p97Eps8 expression was either reduced by TSA treatment or specifically
knockdown by introducing eps8 siRNA (small interference RNA)-expressing
construct into IV5 cells, cell proliferation was significantly inhibited both in vitro and
in vivo.
To date, the involvement of Eps8 in human cancers is still obscure. In our studies,
we observed the overexpression of Eps8 in both human colon cancer cell lines and
human colorectal tumor specimens. Interestingly, as compared to cells with low Eps8
expression (i.e. SW480), elevated FAK expression, serum-induced ERK activation,
and growth rate are detected in cells with high Eps8 expression (i.e. SW620 and
WiDr), implicating the critical role of Eps8 expression in colon cancer formation.
Indeed, reduced cell proliferation in culture and tumor growth in nude mice is
observed in SW620 cells bearing eps8-siRNA. Surprisingly, a statistically strong
correlation between the expression of Eps8 and FAK is also observed in human colon
tumor specimens. In addition, Eps8 knockdown of SW620 cells not only decreased
FAK expression but also reduced Src-mediated FAK phosphorylation on Tyr-861 and
Tyr-397. In agreement with its role in mitogenesis, overexpression of dominant
negative FAK (i.e. FRNK) reduced BrdU incorporation of both SW620 and WiDr
cells and ectopical expression of FAK in cells with eps8-siRNA restored cellular
growth. We thus conclude that Eps8 overexpression in human colon cancer cells
facilitates their proliferation by upregulation and activation of FAK.
Stat3 activation and pathobiology of lung cancer
Wu-Chou Su
Department of Internal Medicine, College of Medicine, National Cheng Kung
University
Lung cancer with its high prevalence, recurrent rate and metastatic potential
becomes the leading cause of cancer death in Taiwan. Malignant pleural effusion
(MPE) is a poor prognostic sign for patients with non-small cell lung cancer (NSCLC).
The generation of MPE is largely regulated by vascular endothelial growth factor
(VEGF), and upregulation of VEGF by Stat3 has been observed in several types of
tumor cells. Stat3, one of the 7 known STAT (Signal Transducers and Activators of
Transcription) family members, is frequently constitutively activated in malignant
cells. Activation of Stat3 is involved in regulating many genes such as cell cycle
progression, cellular proliferation and survival. In this study, we demonstrate
constitutively activated Stat3 in several human lung cancer cell lines and in tumor
cells infiltrated in the pleurae of patients with adenocarcinoma cell lung cancers
(ADCLC) and MPE. The observations suggest that activated Stat3 plays a role in the
pathogenesis of ADCLC. In PC14PE6/AS2 cells, a Stat3-positive human ADCLC cell
line, autocrine IL-6 activated Stat3 via JAKs, not via Src kinase. PC14PE6/AS2 cells
express higher VEGF mRNA and protein than do Stat3-negative
PC14PE6/AS2/dnStat3 cells. In an animal model, PC14P6/AS2/ dnStat3 cells
produced no MPE and less lung metastasis than did PC14P6/AS2 cells.
PC14PE6/AS2 cells also expressed higher VEGF protein, microvessel density, and
vascular permeability in tumors than did PC14P6/AS2/dnStat3 cells. Therefore, we
hypothesize that autocrine IL-6 activation of Stat3 in ADCLC may be involved in the
formation of malignant pleural effusion by upregulating VEGF. Higher levels of IL-6
and VEGF were also found in the pleural fluids of patients with ADCLC than in
patients with congestive heart failure. The autocrine IL-6/Stat3/VEGF signaling
pathway may also be activated in patients with ADCLC and MPE.
Lung cancer cells with constitutively activated Stat3 are more resistant to
cisplatin- and paclitaxel-induced apoptosis. Abrogate the Stat3 signaling by EGCG or
AG490 sensitized cells with activated Stat3 to chemotherapeutic drugs, but provide
survival advantage to cells in which Stat3 was deactivated firstly by transfection with
dn-Stat3. Upon treatment with anti-cancer agents, the activation of Stat3 in
PC14PE6/AS2 cells was enhanced at earlier period (around 3 hours), and then
declined gradually. The further activation of Stat3 in PC14PE6/AS2 cells by
paclitaxel is mediated mainly by mitochondria-respiratory-chain-generated ROS,
which then leads to PKC- and Jak activation.
We have also investigated expression of a group of proteins in the pleural
effusions from lung adenocarcinom, pneumonia and heart failure by ELISA and
identified some proteins with pathological significance. Among them, HER-2/neu and
IGF-1 have the greatest potential. We have shown that HER2/neu and IGF-1 may play
important roles in the pathogenesis of MPE-associated lung adenocarcinoma and may
become ideal tumor markers for pleural effusion diagnosis. In proteomics studies, by
comparing protein expression patterns of MPE from smoking male and non-smoking
female lung cancer patients, we have identified three proteins that are overexpressed
in non-smoking females. They are α-1 inhibitor III precursor, apolipoprotein E
(APOE), and coatomer protein complex, subunit β (COPB). APOE has been
implicated in tumor invasion. By immunohistochemical studies, APOE was found in
the cytoplasm of lung adenocarcinoma cells in patients’ pleurae, suggesting it may
contribute to pathogenesis of the disease. Further studies on the significance of
overexpression of APOE are undergoing. RNA specimens from cancer cells in MPE
were extracted and sent for Affymatrix oligonucleotide microarray analyses. Under
non-supervised clustering analyses, female non-smokers and male smokers were
subdivided into two groups. We also identify a 29-gene-panel that is able to classify
these two groups of patients.
According to the data generated from the current study, Stat3 activation, together
with other oncogenic pathways, contributes to the pathogenesis of MPE formation,
lung metastases, and drug resistance of lung cancer cells.
ER stress signals induced by HBV pre-S mutants: A novel mechanism for viral
carcinogenesis
Hui-Ching Wang, and Ih-Jen Su
Division of Clinical Research, National Health Research Institutes
Hepatitis B viruses (HBV) containing pre-S mutant surface proteins have been
linked to the development of HBV-related hepatocellular carcinoma. In past few years,
our studies have emphasized on potential oncogenic mechanisms of HBV pre-S
mutants. We have identified two major types of pre-S mutants (pre-S1 mutant and
pre-S2 mutant) from patients with chronic HBV infection. The pre-S2 mutant surface
protein is of particular interest in viewing those hepatocytes harboring pre-S2 mutant
proteins usually exhibited clonal proliferation and growth advantages. By double
immunofluorescence staining, we have demonstrated that both pre-S mutant proteins
were localized in the endoplasmic reticulum (ER) as unfolded proteins leading to ER
stress signals. Although ER stress is served as a protective mechanism in response to
stress in the ER, our results revealed that ER stress signal may also involve in viral
carcinogenesis. ER stress induced by pre-S mutant proteins may lead to oxidative
DNA damages and mutagenesis, resulting in genomic instabilities in hepatocytes. In
addition, ER stress can stimulate the expression of cyclooxygenase-2 (COX-2), which
is essential for the anchorage-independent growth of hepatocytes, through the NF-B
and pp38MAPK pathways. Furthermore, pre-S2 mutant protein may enhance cyclin A
expression via its specific transactivator functions. In transgenic mice model, pre-S2
mutant protein could induce nodular proliferation and hyperplasia of hepatocytes,
supporting a contributing role of hepatocarcinogenesis. Recently, we have explored
the oncogenic role of ER stress to the aberrant regulation of centrosome duplication.
We have demonstrated that calpain activation upon ER stress may result in N-terminal
truncation of cyclin A and its cytoplasmic localization, leading to aberrant centrosome
overduplication. Furthermore, our data revealed that pre-S2 mutant protein can induce
hyperphosphorylation of RB tumor suppressor via cyclin dependent kinase-2. The
pre-S2 mutant protein may also impair functions of p27Kip1 through the interaction
with Jun activation domain-binding protein 1 (JAB1). Hence, our results indicated
that ER stress signals may contribute to viral carcinogenesis through the induction of
impaired cell cycle regulation, centrosome overduplication, oxidative DNA damages,
COX-2 expression, RB hyperphosphorylation and degradation of p27Kip1. In
conclusion, a novel mechanism for viral carcinogenesis is provided.
Isolation and characterization of a small size peptide Zfra that regulates
apoptosis by functionally interacting with WOX1
Li-Jin Hsu1, Yee-Shin Lin1 and Nan-Shan Chang2
1
Department of Microbiology and Immunology, National Cheng Kung University
Medical College, Tainan, Taiwan; 2Laboratory of Molecular Immunology, Guthrie
Research Institute, Sayre, Pennsylvania, USA
We isolated an unusual gene transcript encoding a 31-amino-acid zinc finger-like
peptide that regulates apoptosis (named Zfra). Northern blotting and RT/PCR
showed the transcript is abundant in spleen but absent in several prostate and breast
cancer cells. Presence of Zfra peptide was evidenced by in vitro translation,
immunoprecipitation, and isoelectric focusing. Transiently overexpressed Zfra
induced apoptosis, and phosphorylation at Ser8 was essential for its apoptotic function.
Depending upon the extent of expression, Zfra could either enhance or block the
cytotoxic effect of death domain proteins TRADD, FADD and RIP of the TNF and
FasL signaling pathways. TNF and UV light induced physical association of Zfra
with proapoptotic WOX1 (WWOX/FOR2) and its translocation to the mitochondria,
as determined by co-immunoprecipitation, GST pull-down analysis, and
immunofluorescence microscopy. Yeast two-hybrid mapping showed that Zfra
bound to the N-terminal Tyr33-phosphorylated WW domain of WOX1. WOX1 is a
downstream effector of the TNF signaling and capable of enhancing TNF cytotoxicity.
Ectopic Zfra blocked phosphorylation and nuclear translocation of WOX1 and p53
and their apoptotic effects in certain cells. In contrast, a Ser8 mutant of Zfra had no
blocking effect. Together, Zfra is a novel small size peptide that functionally
interacts with downstream effector WOX1 of the TNF and stress pathways.
Fas-triggered
non-death
signaling
pathway
in
tumor
formation
and
autoimmunity
Chung-Chen Su, Yu-Ping Lin, and Bei-Chang Yang
Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung
University
In the first part of this study, we evaluated the Fas signaling in the presence of
tumor/ECM. We demonstrated that the cell contact-induced reduction in the Fas-cross
linking-induced apoptosis in T cells is tumor cell line-dependant. Decreases in the
characteristics of apoptosis including caspase-9, and -3 activation, loss of plasma
asymmetry, cell shrinkage, and loss of mitochondria membrane potential were
observed in Jurkat cells upon coculture with glioma cells, which have the ability to
suppress apoptosis (manuscript in preparation). In following up the dynamic change
of ERK activity, we found that direct cell-cell contact with MCF-7 induced a quick
and transient phosphorylation of ERK in Jurkat T cells. However, prolonged cell
contact reversed the elevated ERK phosphorylation in Jurkat cells in a protein kinase
A (PKA)-dependant way. Furthermore, prolonged contact also enhanced the activity
of ERK-specific phosphatases. We concluded that T cell chemokines, will activate
RAF/MEK/ERK in T cells. However, prolonged integrin activation by ECM
engagement increased cAMP level and activated PKA, which subsequently
phosphorylated the Raf at 259 a.a. site leading to inhibition of Raf activity and shut
down the MEK/ERK. These findings suggest that anchorage on ECM of tumor can
dynamically regulate T cell recruitment.
Second, we found that Fas signaling intrinsically activated p38 MAPK pathway
by itself to suppress caspase-cascade, which in turn reduced cell death. Inactivation of
caspase 8 by Z-IETD eliminated the phosphorylation of p38MAPK in response to Fas
treatment. Moreover, phosphor-p38 (pp38) is colocalized with caspase 8. The
expression of pp38, reflecting the activity of p38 kinase, was higher in synovial T
cells from Rheumatoid arthritis (RA) as compared with that in T cells isolated from
normal peripheral blood. Moreover, synovial fluid stimulated the p38 in T cells
implicate a role of this kinase in RA (manuscript in preparation). In addition to the
novel Fas pathway regulating cell death, we further examined whether caspase-3 may
stimulate tumor metastasis. When caspase-3 was introduced into the MCF-7 breast
cancer cell line, which was originally caspase-3 deficient, cell motility and lung
metastasis were significantly enhanced. In parallel, the caspase-3 expression of A549
lung carcinoma cells was reduced by RNA interference (RNAi) strategy. MCF-7 cells
overexpressing caspase-3 did not show significant traits of apoptosis. In addition, the
caspase-3 overexpressing MCF-7 cells possessed higher motility and secreted more
MMP-2. On the contrary, caspase-3 siRNA reduced motility and invasiveness of A549
cells. By using the in vivo experimental lung metastasis model, we observed that
caspase-3 increased the severity of metastasis. In both MCF-7 and A549 cells, we
observed a caspase-3 mediated extracellular signal-regulated kinase (ERK) activation,
which is independent of caspase-3 activity. In addition, Erk phosphorylation is
dominant in caspase-3 mediated cell migration enhancement. Inhibition of Erk
activation strongly decreased cell motility. Collectively, we demonstrated that caspase
3 might involve in tumor metastasis (Manuscript in preparation).
Low substratum rigidity suppresses FAK397 phosphorylation, but augments
Erk1/2 phosphorylation to trigger cell migration
Yu-Chih Hsu, Wei-Chun Wei, and Ming-Jer Tang
Department of Physiology, College of Medicine, National Cheng Kung University
Previous study demonstrated that cells cultured on collagen gel displayed
down-regulation of focal adhesion proteins induced by low rigidity. In this study, we
found that cells cultured on collagen gel exhibited higher migration capacity than
those cultured on collagen gel-coated dishes, suggesting that low substratum rigidity
triggers cell migration. Cells cultured on collagen gel displayed enhanced FAK
tyrosine phosphorylation similarly with those cultured on collagen gel-coated dishes,
with exception that FAKY397 phosphorylation was spared. In contrast, low rigidity
induced delayed but persistent phosphorylation of ERK1/2. Inhibition of collagen
gel-induced ERK1/2 phosphorylation by MEK inhibitors and ERK2 kinase mutant
resulted in cell round up and reduction of collagen gel-induced cell migration.
Interestingly, the collagen gel-induced ERK1/2 phosphorylation was present in focal
adhesion site, indicating that ERK1/2 activation leads to cell spreading and migration.
Moreover, MCD (Methyl-cyclodextrin), lipid raft/caveolae inhibitors, prevented
collagen gel-induced phosphorylation of ERK1/2 as well as cell migration. MDCK
cells overexpressing FAKY397F mutant exhibited higher levels of ERK
phosphorylation than those overexpressing wild type FAK. Taken together, low
rigidity induces lipid raft-mediated activation of ERK1/2, which is present in focal
adhesion to facilitate cell spreading and migration.
Molecular mechanism of ceramide-induced apoptosis: the anti-apoptotic role of
lithium
Yee-Shin Lin, Chiou-Feng Lin, Chia-Ling Chen, Ming-Shiou Jan, Li-Jin Hsu, Peng-Ju
Chien, and Chi-Wu Chiang
Department of Microbiology and Immunology, College of Medicine, National Cheng
Kung University
Ceramide, a product of sphingolipid metabolism functions as intracellular second
messenger, may modulate the biochemical and cellular processes in response to
various apoptotic stress stimuli. However, the signaling pathways of
ceramide-induced apoptosis are not fully defined. Our previous studies showed that
sequential activation of caspase-2 and -8 is essential for ceramide-induced
mitochondrial apoptosis. Bcl-2 rescues ceramide-induced apoptosis through blockage
of caspase-2 activation. Protein phosphatase 2A (PP2A)-mediated Bcl-2
dephosphorylation is involved in caspase-2 activation induced by ceramide.
Furthermore, lithium conferred protection against ceramide-induced apoptosis by
activation of MEK/ERK and inhibition of caspase-2 and -8. We recently showed that
lithium blocked ceramide-induced apoptosis via inhibition of PP2A activity.
Ceramide-induced PP2A activation involved methylation of PP2A C subunit, which
lithium suppressed. Lithium caused dissociation of PP2A B subunit from the PP2A
core enzyme, whereas ceramide caused recruitment of the B subunit. Further study
showed a role of glycogen synthase kinase-3 (GSK-3 in ceramide-induced
apoptosis, in which GSK-3 acted downstream of PP2A and PI3K/Akt and upstream
of caspase-2 and caspase-8. In addition to a direct effect of lithium on GSK-3 as
previous known, our study showed that lithium may cause GSK-3 phosphorylation
and inactivation through PI3K/PKB- but not ERK-mediated pathway. Microarray
analysis showed that ceramide upregulated thioredoxin-interacting protein (TXNIP)
expression, whereas lithium downregulated its expression. The involvement of
TXNIP in p38 MAPK- and JNK-mediated apoptotic signaling pathways was
demonstrated. Interestingly, our recent results showed the involvement of acid
sphingomyelinase and ceramide in spontaneous thymocyte apoptosis and lithium may
prevent cell death by inhibiting GSK-3 and caspase-2 activation. Taken together, our
studies show both transcription-independent and transcription-dependent pathways of
ceramide-induced apoptotic cell death, and lithium confers an anti-apoptotic effect in
both pathways.