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Transcriptional mechanism of Sp1-mediated genes in A431 cells
Jan-Jong Hung, Yi-Ting Wang and Wen-Chang Chang
Department of Pharmacology, College of Medicine, National Cheng Kung University
Two parts in our study are carried out to characterize the transcriptional mechanism of
Sp1-mediated genes. First, 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. In this study, we found in vivo and in vitro that Hsp90 was recruited to the
GC-rich region of 12(S)-lipoxygenase promoter by interaction with Sp1 in A431 cells
by
employing
DNA
affinity
immunoprecipitation
assay
and
chromatin
immunoprecipitation assay. When Hsp90 was inhibited by geldanamycin (GA), a
specific inhibitor of Hsp90 family, or by siRNA of Hsp90 to block its activity or to
knockdown protein level respectively, the luciferase activity driven by the
12(S)-lipoxygenase promoter, and both of mRNA and protein levels of
12(S)-lipoxygenase all were reduced significantly in cells. In addition, the effect of
GA was abolished when the Sp1-binding sites of 12(S)-lipoxygenase were mutated in
A431 cells. Interestingly, binding of Sp1 to the 12(S)-lipoxygenase promoter was also
decreased upon GA treatment in cells. In conclusion, our results indicated that Sp1
interacted with Hsp90 to recruit it to the promoter of 12(S)-lipoxygenase, and then to
regulate the gene transcription by affecting the binding ability of Sp1 to the promoters.
Second, we previous reported that Sp1 functions as an anchor to recruit c-Jun to the
promoter of the 12(S)-lipoxygenase gene when human epidermoid carcinoma A431
cells were stimulated with 12-myristate 13-acetate (PMA) or epidermal growth factor.
We now show that Sp1 was constitutively acetylated that recruited HDAC1 to the
Sp1/cJun complex when cells were exposed to PMA (3 h). Prolonged stimulation of
the cells with PMA (9 h), however, caused the dissociation of HDAC1 and the
deacetylation of Sp1 with the latter being able to recruit p300 that in turn caused the
acetylation and dissociation of histone 3, thus enhancing the expression of
12(S)-lipoxygenase. To establish a causal relation between the decetylation of Sp1
and the recruitment of p300, we overexpressed Sp1 mutant (K703/A; lacking
acetylation sites) in the cell and found that mutant cells recruited more p300 and
expressed more 12(S)-lipoxygenase. Our results indicate that the activation of a
promoter may require several transcription factors acting in a highly coordinated,
temporal-spatial manner. Our results also suggest that transcription factors, as
limited in numbers as they are found in nature, can perform formidable arrays of gene
regulation in mammals perhaps by landing numerous combinations and/or temporal
spatial arrangements at even a single locus on a promoter.
The regulatory mechanisms of increased p300 recruitment to the keratin 16
promoter by ERK activation in keratinocytes
Ying-Nai Wang, Yun-Ju Chen and Wen-Chang Chang
Department of Pharmacology, College of Medicine, National Cheng Kung University
In studies of the transcriptional regulation of keratin 16, we have provided a proposed
model, indicating that Sp1 recruits c-Jun to the promoter through the binding of Sp1
to the Sp1 site, and coactivators p300/CBP interact with Sp1 and AP1 proteins and
participate in the transcriptional regulation for EGF induction of keratin 16 gene
expression in keratinocyte HaCaT cells. By using a chromatin immunoprecipitation
assay and a DNA affinity precipitation assay, EGF treatment up-regulates p300
recruitment through ERK signaling to the promoter region in regulating keratin 16
transcriptional activity. p300 mediates and regulates EGF-induced keratin 16 gene
expression at least in part through multiple mechanisms including a selective
acetylation of c-Jun and histone H3. It was reported previously that direct
phosphorylation of the coactivator CBP represents a novel mechanism controlling its
recruitment to the transcription complex. Furthermore, the documents were found that
the transcriptional activity of p300 was stimulated by phenylephrine through p42/p44
MAPK cascade, and the transactivation domain (aa 1572-2370) of p300 was
phosphorylated by MAPK in vitro. According to our results, p300 was phosphorylated
by ERK activation in vitro and in vivo. However, it is still unclear where the
phosphorylation sites occur upon growth factors treatment. More importantly, the
functional links between the specific phosphorylation events and the downstream
gene regulations remain largely unknown. Whether recruitment of p300 to the keratin
16 promoter by ERK activation was regulated by its phosphorylation state will be
further explored. In addition, it has been reported that p300 acts as a scaffold for the
cooperation with multiple transcriptional factors such as Sp1. Nevertheless, it remains
unknown whether any post-translational modification of Sp1 by EGF treatment occurs
and then promotes keratin 16 gene activation by protein-protein interaction with p300.
In our system, p300 could interact with Sp1 by using GST-pull down assay in vitro,
and a significant binding of co-immunoprecipitated p300 to Sp1 was also observed in
cells upon EGF stimulation in vivo. Taken these results together, the enhanced binding
of p300 to Sp1 by EGF treatment could be mediated by phosphorylation of p300
through ERK. Therefore, we are interested to further clarify the underlying regulatory
mechanisms, such as through physical protein-protein interaction and posttranslational modification, of those nuclear factors involved in the transcriptional
control of keratin 16 upon EGF treatment.
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
c-Jun/Sp1 interaction. PP2B bound and dephosphorylated the phospho-TAM-67
interacted with c-Jun in PMA-treated cells. To investigate the function
mechanism involved in the regulation of c-Jun/PP2B complex, EGF-
and
and
and
and
PMA-induced protein modification was analyzed. EGF induced GSK3
phosphorylation but no change in GSK3/Sp1 complex. PMA but not EGF induced
MAPK phosphorylation was inhibited in cells treated with PP2B, HDAC and GSK3
inhibitors. Theses results suggested that c-Jun/PP2B may play different function in
EGF- and PMA-induced gene expression.
Regulation of gene expression by the VDR/Sp1 complex
Yu-Chun Huang, Jin-Yi Chen, Hsuen-Tsen Cheng 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 activate gene expression. 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. 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. We also identify that VDR/Sp1 complex
can activate CD14 via similar mechanism. Therefore, p27Kip1 and CD14 are molecular
targets that positively regulated by the VDR/Sp1 complex. Microarray analysis
identifies several potential target genes which expression may be inhibited by the
VDR/Sp1 complex. 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. Taken together, our
study suggests that VDR may form the VDR/Sp1 complex and modulate gene
expression via the Sp1 site in the promoter.
Post-transcriptional regulation of thrombomodulin
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
showes 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 EGF-induced upregulation of TM mRNA in human cervical cancer
cell line A431 is accompanied by stabilization (around 2-fold) of TM mRNA. 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, four major binding
proteins HuR, poly(C)-binding protein (PCBP), heterogeneous nuclear
ribonucleoprotein K (hnRNP-K) and polypyrimidine tract-binding protein 1 (PTBP1
or hnRNP-I) were identified by UV-crosslinking assay and mass spectrometry.
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.
The mechanism for nuclear translocalization of RON receptor 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
RON (Recepteur d’Origine Nantais) is a distinct receptor tyrosine kinase in the MET
proto-oncogene family. A number of studies have described the potential significance
of RON overexpression in epithelial carcinomas. We recently demonstrated that
MSP/RON-associated signaling is important in the progression of human bladder
cancer. Intriguingly, both wild-typed and truncated forms of RON were observed in
the nuclei. This study was thus designed to explore the molecular basis underlying the
nuclear translocalization of RON in human bladder carcinogenesis. Both wild-typed
and truncated forms of RON were demonstrated in the nuclear fraction of cancer cells
at de-phosphorylated status via a ligand-independent manner. Nuclear RON was also
colocalized with importin 1, 1 and de-phosphorylated EGFR. However MSP
treatment resulted in phosphorylation of EGFR accompanied by dissociation from
RON in the nuclei, implying a cross-talk between RON and EGFR. The siRNA
experiments confirmed the importance of EGFR for nuclear translocalization of RON.
Then site-directed mutagenesis assay on N- and/or C-terminal nuclear localization
signals (NLSs) of RON was performed to clarify the significance of NLS in the
nuclear translocalization of RON. Preliminary results showed that R306Q at
N-terminal, and both R1388T and R1389T at C-terminal NLS of RON effectively
suppressed the nuclear translocalization of RON, supporting the involvement of NLS
in the nuclear localization of the receptor protein. Moreover, both proliferation and
anti-apoptotic effects were suppressed in NLS mutants compared with wild-typed
transfectants, suggesting the importance of nuclear RON in the cell proliferation and
anti-apoptosis. Given that heat shock proteins (HSPs) were shown to be the
chaperones in regulating stress-induced signaling and nuclear transport of cell cycle
kinases, we assayed their expression in the subcellular fraction by immunoblotting.
Levels of HSP70, HSP90 and heat shock factor 1 (HSF 1) were all increased in the
cell nuclei. Further study is underway to examine their biological significance for
nuclear RON/EGFR complex in human bladder cancer.
Eps8 regulates Src/FAK activity in colorectal cancer cells
Tzeng-Horng Leu1, Ming-Chei Maa2, Jenq-Chang Lee3, Yun-Ju Chen1, Yen-Jen Chen1,
Ching-Chung Huang1, Shan-Tair Wang4, Nan-Haw Chow5 and Yuan Liu1
1
Department of Pharmacology, 3Surgery, 4 Public Health, and 5 Pathology, College of
Medicine, National Cheng Kung University, 2 Institute of Biochemistry, Chung Shan
Medical University
Eps8, a common substrate for both receptor and nonreceptor tyrosine kinases, has
been characterized as an oncoprotein and participates in Src-mediated transformation
in murine fibroblasts. However, to date, its involvement in human cancers is still
obscure. In this study, we observe 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 and HCT116), elevated FAK
expression, 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-863 and –577, resulting in the decrease of FAK Tyr-397
phosphorylation. 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.
The study of mechanisms by which paclitaxel activates Stat3 protein in lung
cancer cells
Ya-Chin Lo, I-Huei Tu, Hsuan-Heng Yeh and Wu-Chou Su
Department of Internal Medicine, College of Medicine, National Cheng Kung
University
Stat3, one of the 7 known STAT (Signal Transducers and Activators of Transcription)
family members, is frequently constitutively activated in malignant cells1. Activation
of Stat3 is involved in regulating many genes such as cell cycle progression, cellular
proliferation and survival. In our system, constitutively activated Stat3 in human lung
adenocarcinoma cell line --PC14PE6/AS2, mediated by IL6/gp130/Jak2 pathway, was
demonstrated. 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 could be abolished by adding Jak2 inhibitor – AG490 – indicates that Jak2
mediates the reaction. For the mechanisms underlying the activation of Jak2 by
anticancer agents, we suspect the induction of ROS and subsequent deactivation of
tyrosine phosphatase may play a role. Using the redox-sensitive fluorescence probe
DCFH-DA, we found that anti-cancer agent, paclitaxel, stimulates ROS production in
PC14PE6/AS2 cells. Generation of ROS was suppressed by diphenylene iodonium
(DPI), an inhibitor of flavoprotein-dependent oxidases, Rotenone, an inhibitor of
mitochondrial respiratory chain complex I, and Antimycin A, an inhibitor of
mitochondrial respiratory chain complex III. The activation of Stat3 in PC14PE6/AS2
cells by paclitaxel is also inhibited by the pretreatment of the above three compounds.
Other inhibitors of NAC or catalase did not abolish Stat3 activation by paclitaxel.
Taken together, paclitaxel induces ROS generation is likely through a
mitochondrial-mediated pathway. In the other part, we suspect that generation of ROS
by paclitaxel may transiently inactivate PTP-1B and then switch the equilibrium
towards autophosphorylation of Jak kinases. Although this phenomenon was not
observed, we found another pathway may partially involved STAT3 activation
induced by paclitaxel. H2O2-induced activation of PKC- is reported to be
independent from tyrosine phosphatase inhibition. Inhibition of PKC-
rottlerin supported that PKC- kinase activity is required for both baseline and
paclitaxel-induced STAT3 tyrosine phosphorylation. The paclitaxel-induced STAT3
activation resulted in upregulation of Bcl-2 protein, which may contribute to the
relatively resistant to paclitaxel in PC14PE6/AS2 than in A549 cells. With a better
understanding of how anticancer agents regulate Stat3 activation, we may have
chances to find new targets for overcoming tumor drug resistance.
HBV pre-S2 mutant and endoplasmic reticulum stress signaling pathways
Yung-Mei Chao, Yung-Sheng Chang, Ih-Jen Su, Huan-Yao Lei, Wen-Tsan Chang,
Wenya Huang, Jui-Hsiung Huang, Hui-Ching Wang, Wen-Chang Chang and
Ming-Derg Lai
Department of Biochemistry and Department of Basic Medicine, College of Medicine,
National Cheng Kung University
The expression of HBV pre-S2 mutant has been associated with the development of
hepatocarcinoma. Endoplasmic reticulum stress induced by HBV pre-S2 mutant may
play a role in carcinogenesis and genomic instability. In this report, we investigated
the possible signal pathways resulting from the HBV pre-S2 mutant or endoplasmic
reticulum stress. Since c-Jun is important for hepatocyte development, we first studied
whether there is a linkage between HBV pre-S2 mutant, ER stress, and c-Jun.
Tunicamycin, and brefeldin A, two ER stress inducers, increased the expression of
c-Jun in ML-1 cells. Expression of HBV pre-S2 mutant also enhanced the expression
of c-Jun in ML-1 cells. Furthermore, one of the c-Jun-regulated genes, MMP-9 was
induced under ER stress and the expression of HBV-large surface proteins. Enhanced
binding of c-Jun on the promoter of MMP-9 gene was demonstrated by EMSA and
CHIP. In addition, the migration ability of ML-1 cells expressing HBV pre-S2 mutant
was higher than that of ML-1 cells expressing wild-type HBV large surface proteins.
In the future, our laboratory will focus on several interesting topics: (1) what is the
mechanism of enhanced migration in cells expressing HBV pre-S2 mutant? (2) Does
lipid biosynthesis play a role in causing carcinogenesis? (3) How does HBV pre-S2
affects the transcriptional factor and coactivator through ER stress?
The role of HBV pre-S2 mutant protein in hepatocarcinogenesis
Hui-Ching Wang, Jui-Chu Yang and Ih-Jen Su
Division of Clinical Research, National Health Research Institutes, Tainan
Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung
University
In past few years, we have demonstrated that pre-S2 mutant protein (S2-LHBs)
could serve as a tumor promoter that contributes to carcinogenesis via either gene
transactivation or ER stress signaling. The inductions of cyclin A in both liver tissues
and transgenic mice by S2-LHBs have shown biological impacts on liver nodular
proliferation and hepatocyte multinucleation. Since gene expression of cyclin A is
regulated by RB/E2F pathway during G1 phase of cell cycle, it is important to
evaluate whether this signal pathway is affected by S2-LHBs protein. On HuH-7
cells, hyperphosphorylated form of RB is increased by S2-LHBs as demonstrated by
Western Blotting. Although the exact phosphorylation site on RB induced by
S2-LHBs remains further examination, we have evidence showing that DNA
cytosine-5 methyltransferase 1 (DNMT1) may be induced by the release of E2F1
from RB. Since DNMT1 is required in maintaining DNA methylation in both normal
and cancer cells, the induction of DNMT1 may represent an epigenetic cause of
cancer in the development of hepatocellular carcinoma. Recently, we have also
demonstrated that S2-LHBs can induce tumor formation on the transgenic mice
model when established on the genetic background of C57/B6 strain. The successfully
induction of liver tumors on transgenic mice may therefore provide us more
information to study the carcinogenic pathway involving in HBV pre-S mutants
during the development of hepatocellular carcinoma.
Characterization of a small size peptide Zfra that regulates stress responses by
functionally interacting with WOX1 and JNK1
Li-Jin Hsu1, Yee-Shin Lin1 and Nan-Shan Chang2
1
Department of Microbiology and Immunology, College of Medicine, National Cheng
Kung University, Tainan, Taiwan; 2Laboratory of Molecular Immunology, Guthrie
Research Institute, Sayre, Pennsylvania, USA
Here, we isolated an unusual gene transcript encoding a 31-amino-acid zinc
finger-like protein or 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 protein was evidenced by in vitro
translation, immunoprecipitation, and isoelectric focusing.
Transiently
overexpressed Zfra induced apoptosis, and that phosphorylation of Ser8 is 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. Mechanistically, TNF and UV
light induce Zfra to rapidly selfassociate and bind WOX1, JNK1 and NF-B (p65).
The tumor suppressor p53 weakly interacted with Zfra. Ectopic Zfra blocks
phosphorylation and nuclear translocation of WOX1, JNK1 and p53 and their
apoptotic effects in certain cells. In contrast, a Ser8 mutant of Zfra has no blocking
effect.
Together, Zfra is a novel small size peptide that functionally interacts with
downstream effectors WOX1, JNK1, p53 and NF-B of the TNF and stress pathways.
Discussion for the Fas-mediated apoptosis of immune T cell reduced by tumor
cell
Chung-Chen Su, Yu-Ping Lin and Bei-Chang Yang
Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung
University
FasL expression on tumor cells engaged with the Fas protein of tumor infiltrating
lymphocytes (TILs) will trigger death signal to lead these cells into apoptosis. We
established a model system to utilize immune cells cocultured with tumor cells and
simulated the specific environment of tumor nodule to investigate the affection of
ECM or adhesion molecular on Fas-mediated apoptosis of TILs. As human Jurkat T
cells were cocultured with glioma through cell-cell direct contact, the sensitivity of
apoptosis in Jurkat T cells induced by CH-11 (anti-human Fas monoclonal antibody)
was reduced. The biochemical activities involved in apoptosis, such as activation of
caspase-8, -9, or –3 and DNA fragmentation are reduced by cocultured with glioma
cells. In addition, tumor cells will reduce the activation-induced cell death process of
activated peripheral T cell in blood. Furthermore, kinases activity of immune cells can
be activated through the interaction of ECM and integrins. Our previous result
showed that phosphorylation of Erk 1/2 and p38 MAPK activated by treating CH-11
alone or cocultured with glioma cells but inhibition of Erk 1/2 or p38 MAPK
phosphorylation does not affect the reduction of CH-11-induced cell death in Jurkat T
cells by glioma. CH-11 reduces the AKT phosphorylation of Jurkat T cells, but
recover to basal level under cocultured with glioma. Recently, we found that
phosphorylation of Erk 1/2, p38 MAPK and JNK were strong enhanced but the
CH-11-induced cell death of Jurkat T cells did not reduce when MCF-7 existence.
Altogether, the roles of kinase activity play in immune cells through cell-cell direct
contact to reduce the Fas-mediated death signaling transduction remain to be clarified.
Second part of the project showed that engagement of Fas with CH-11 induced the
phosphorylation of p38 MAPK in Jurkat T cells. No new protein synthesis was
required for the Fas-mediated phosphorylation of p38 MAPK. Inactivation of
caspase-8 by specific inhibitor Z-IETD reduced the phosphorylation of p38 MAPK
induced by CH-11. Suppression of p38 MAPK activity significantly enhanced the
Fas-mediated apoptosis. Moreover, Fas-associated caspase-8 and caspase-3 induction
were enhanced by the inhibition of p38 MAPK. The enhanced activation of caspase-3
upon Fas signaling by SB202190 was completely blocked by Fas antagonistic
antibody ZB4 or caspase-8 inhibitor Z-IETD indicating that the influence of p38
MAPK on caspase-3 was specific in Fas signaling. The involvement of p38 MAPK in
Fas-mediated apoptosis was also observed in peripheral activated-T cells. The
expression of pP38 in fresh T cells and non-activated T cells were more than
activated-T cells supporting that containing more pP38 may be resistance to
Fas-mediated apoptosis. In summary, the activation of p38 MAPK by Fas signal was
an auto feedback pathway that prevents the death event in T cells.
Low substratum rigidity down-regulates FAK397 phosphorylation and 1 integrin
by different ways
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 when MDCK cells were cultured on collagen gel, the FAK397
phosphorylation ratio was decreased while other phosphorylation sites (407, 577, 861,
and 925) of FAK remained activated. We also checked the level of 1 integrin
activation which is the upstream signal of FAK. 1 integrin activation was
down-regulated under low substratum rigidity. To analyze whether FAK and DDR1,
another collagen receptor, were involved in the “in-side-out” signal of 1 integrin
activation, wild type and dominant negative FAK or DDR1 stably transfected MDCK
cells were employed. We found that low rigidity induced down-regulation of 1
integrin activation and FAK397 phosphorylation was not altered by FAK or DDR1. To
elucidate whether the internal force provided from actin filaments and microtubules
affected 1 integrin activation and FAK397 phosphorylation, we employed
cytochalasin D and colcemide. Disruption of actin cytoskeleton by cytochalasin D
blocked FAK397 phosphorylation but not 1 integrin activation while disruption of
microtubules by colcemide had no effect. Immunofluorescence showed that active 1
integrin was colocalized with lipid raft in cells cultured on collagen-coated dish.
Furthermore, MCD, a lipid raft inhibitor, inhibited 1 integrin activation in cells
cultured on rigid substratum. However, MCD did not affect the level of FAK397
phosphorylation. Taken together, our data provides a new concept that collagen
fibril-induced FAK397 phosphorylation needs internal force provided from actin
filaments. On the other hand, 1 integrin activation requires preferentially external
force from rigid substratum, which is regulated by lipid raft.
ER stress is involved in apoptosis induced by low substratum rigidity
Wen-Tai Chiu1, Meng-Ru Shen2, 3, Yao-Hsien Wang1 and Ming-Jer Tang1, 4
1
Institute of Basic Medical Sciences, 2Department of Pharmacology, 3Department of
Obstetrics & Gynecology, 4Department of Physiology, College of Medicine, National
Cheng Kung University
Mechanical probing of the immediate environment is considered a critical mechanism
controlling several cellular processes, such as motility, morphogenesis, proliferation,
and apoptosis. Our previous studies have showed that collagen gel induced apoptosis
in epithelial cells, but not in mesenchymal cells and tumor cells. This kind of cell
apoptosis was mediated by the physical property of low-substratum rigidity, but the
mechanisms that cells senses the substratum rigidity remain poorly understood. Here,
we investigated the underlying mechanism of low-substratum rigidity induced
apoptosis. ER stress-specific caspase-12 and effector caspase-3 were activated in
LLC-PK1 cells when cultured on collagen gel. Down-regulation of ER-resident Ca2+
buffering proteins (calregulin and calnexin) and activation of calcium-activated
cysteine protease (-calpain) were also noted. Collagen gel also caused ER-Ca2+
overloading which subsequently up-regulated capacitative calcium entry. Both CCE
inhibitors (BEL and 2-APB) and intracellular Ca2+ chelator (BAPTA-AM) could
inhibit significantly collagen gel-induced -calpain activation and apoptosis in
LLC-PK1 cells. This indicates that the disturbance of intracellular Ca2+ homeostasis
likely contributes to ER stress leading to low-substratum rigidity induced apoptosis.
In conclusion, collagen gel-induced ER stress is involved in low substratum
rigidity-induced epithelial cell apoptosis. Dysregulation of Ca2+ homeostasis may play
an important role in this phenomenon.
Deregulation of AP-1 protein family in collagen gel–induced apoptosis mediated
by low substratum rigidity
Yao-Hsien Wang1, Wen-Tai Chiu1, Yang-Kao Wang2, Pei-Jun Hsieh2 and Ming-Jer
Tang1, 2
1
Institute of Basic Medical Sciences, 2Department of Physiology, College of Medicine,
National Cheng Kung University
In order to delineate how the substratum rigidity controlled cell life and death, we
employed cell cultures on type I collagen gel or collagen gel-coated dish. Here we
established that collagen gel, but not collagen gel-coating, induced apoptosis of
epithelial (NMuMG, BS-C-1, LLC-PK1, NRK-52E and BAEC) but not mesenchymal
(HEK 293 and NIH-3T3) or tumor (HK-2, U-373MG, OC-2, DOK, SSC-25, HSC-3
and Chang Liver) cell lines, indicating that low substratum rigidity may be the cause
of apoptosis. To delineate whether rigidity of collagen gel controlled epithelial cell
apoptosis, we employed collagen gels harboring different rigidity due to cross-linking
or physical disruption of collagen fibrils. We found that collagen gel prepared from
older rat ameliorated contracted morphology and apoptosis. On the other hand, a
reduction in rigidity by sonication of collagen fibrils augmented collagen gel-induced
apoptosis. As assessed by rheometry, the rigidity of collagen gel, ranged from 10 to
120 Pascal, was elevated by age effects and lowered by sonication. Bcl-2
overexpression did not prevent low rigidity-induced apoptosis and mitochondria
release of cytochrome c was not observed during cell apoptosis, indicating that the
mitochondrial pathway is not involved in low rigidity-induced apoptosis. Low rigidity
triggered activation of JNK within 4 h. In addition, low rigidity induced rapid
down-regulation of c-Jun, a downstream signal protein of JNK, within 1 h and
triggered aberrant expression of c-Fos, which lasted for at least 24 h. Both reduction
of c-Jun expression by si-RNA and overexpression of c-Fos could induce apoptosis in
several epithelial cells. Inhibition of low rigidity-induced JNK activation by
SP600125 could prevent aberrant c-Fos expression, but only partially blocked low
rigidity-induced apoptosis. Taken together, we conclude that low substratum rigidity
induces apoptosis in epithelial cells, which is mediated at least in part by deregulation
of AP-1 proteins.
Molecular mechanism of ceramide-induced apoptosis and 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 in response to various stress stimuli, such as tumor necrosis factor-, Fas,
chemotherapeutic agents, and irradiation. Ceramide may modulate the biochemical
and cellular processes that lead to apoptosis. However, the mechanisms by which
ceramide regulates apoptotic events are not fully defined. We recently show 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. Our
previous results showed that lithium conferred protection against ceramide-induced
apoptosis by promoting MEK/ERK and inhibiting caspase-2 and -8 activation.
Furthermore, lithium blocked ceramide-induced apoptosis via inhibition of PP2A
activity. Ceramide-induced PP2A activation involved methylation of PP2A C subunit,
which was inhibited by lithium. Lithium caused dissociation of the PP2A B subunit
from the PP2A core enzyme, whereas ceramide caused recruitment of the B subunit.
Further study showed the dependence of GSK-3 in ceramide-induced mitochondrial
apoptosis. The PP2A-regulated PI3K/Akt signaling was involved in GSK-3
activation by ceramide, which lithium suppressed. Microarray analysis showed that
ceramide upregulated thioredoxin-interacting protein (TXNIP) expression, whereas
lithium downregulated its expression. The preliminary results suggest the involvement
of TXNIP in p38- and JNK-mediated apoptotic signaling pathways. 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.