Download c-Jun/Sp1 interaction is essential for growth factor

Calcineurin-mediated dephosphorylation of c-Jun C-terminus is required for
c-Jun/Sp1 interaction in phorbol ester-induced 12(S)-lipoxygenase 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 ester-induced
expression of genes, including human 12(S)-lipoxygenase. This study examined the
mechanism used to regulate the interaction between c-Jun and Sp1 in the phorbol
12-myristate 13-acetate (PMA)-induced expression of 12(S)-lipoxygenase in human
epidermoid carcinoma A431 cells. Treatment of cells with PMA induced
dephosphorylation of c-Jun C-terminus. PMA-induced promoter and enzymatic
activities of 12(S)-lipoxygenase were dose-dependently inhibited by a specific
calcineurin (PP2B) inhibitor, cyclosporin A. Overexpression of PP2B also enhanced
the promoter activity of the 12(S)-lipoxygenase gene in a dose-dependent manner.
Moreover, in PMA-treated cells, cyclosporin A inhibited the interaction between c-Jun
and Sp1. Overexpression of TAM-67, an N-terminally truncated c-Jun, inhibited the
PMA-induced promoter activity of 12(S)-lipoxygenase. We used TAM-67-M3, an
expression vector of a mutant of TAM-67, to test the effect of phosphorylation and
dephosphorylation of the C-terminus of c-Jun on its interaction with Sp1.
TAM-67-M3, which contains three substitutive alanines at Thr-231, Ser-243, and
Ser-249, was about twice as efficacious as TAM-67 in inhibiting the PMA-induced
promoter activity of the 12(S)-lipoxygenase gene. Coimmunoprecipitation
experiments indicated that, compared to TAM-67, TAM-67-M3 bound more
efficaciously with Sp1. Further, cyclosporin A inhibited the PP2B-induced
enhancement of the interaction between TAM-67 and Sp1. In order to verify
PMA-induced dephosphorylation of c-Jun C-terminus was mediated by PP2B, PP2B
siRNA expression vector pSUPER-PP2B-A was used. Overexpression of
pSUPER-PP2B-A in cells caused knockdown of endogenous PP2B and resulted in
reducing the effect of PMA-induced promoter activity and c-Jun/Sp1 interaction.
Moreover, coimmunoprecipitation experiments indicated that PMA could induce
interaction between c-Jun and PP2B. GSK3 induced phosphorylation of GST-TAM-67
was also reduced by PP2B in in vitro kinase assay. Taken together, these results
indicate that PP2B plays an important role in regulating c-Jun/Sp1 interaction in
PMA-induced expression of 12(S)-lipoxygenase. Specifically, the dephosphorylation
of Thr-231, Ser-243, and Ser-249 at the c-Jun C-terminus is essential for the
protein-protein interaction between c-Jun and Sp1.
Activation of MAP kinase by epidermal growth factor mediates c-Jun activation
and p300 recruitment in keratin 16 gene expression
Ying-Nai Wang and Wen-Chang Chang
Department of Pharmacology, College of Medicine, National Cheng Kung University
Overexpression of keratin 16 has been observed in keratinocytes in those skin
diseases characterized by hyperproliferation such as psoriasis. In studies of gene
regulation of keratin 16, we reported previously that Sp1 shows a functional
cooperation with c-Jun and coactivators p300/CBP in driving the transcriptional
regulation of EGF-induced keratin 16 gene expression (Wang and Chang, J. Biol.
Chem. 278, 45848-45857, 2003). In this study, we explored the signaling networks
relaying EGF stimulation of keratin 16 gene expression and the underlying regulatory
mechanism, such as post-translational modification, of those nuclear factors involved
in the transcriptional control of keratin 16 upon EGF treatment. Evidence obtained
from the present study showed that treatment of HaCaT cells with EGF resulted in a
rapid phosphorylation of ERK and JNK, and subsequent induction of c-Jun. The
stimulated expression of keratin 16 by EGF was mediated mainly through the
Ras-MEK-ERK signaling pathway, but partly through the JNK cascade. On the other
hand, Ser63 and Ser73 on the c-Jun NH2-terminal transactivation domain could be
phosphorylated in HaCaT cells treated with EGF. Nevertheless, we found surprisingly
that the c-Jun NH2-terminus was not required for EGF-induced expression of keratin
16. The activation of keratin 16 promoter by EGF treatment could not be enhanced by
overexpression of myc-c-JunK3R, in which three putative acetylation lysine residues
on the c-Jun COOH-terminus were all mutated into arginines, indicating that c-Jun
acetylation might partially play a functional role in this system. Interestingly, by using
a chromatin immunoprecipitation assay and a DNA affinity precipitation assay, EGF
treatment was found to up-regulate p300 recruitment through ERK signaling to the
promoter region in regulating keratin 16 transcriptional activity. In conclusion, these
results strongly suggest that c-Jun biosynthesis, stimulated through ERK and JNK
activation, plays an essential role in regulating keratin 16 gene expression by EGF.
Recruitment of p300 to the keratin 16 gene promoter was due to EGF-activation of the
ERK signaling pathway, and p300 mediated and regulated EGF-induced keratin 16
gene expression, at least in part through multiple mechanisms including a selective
acetylation of c-Jun COOH-terminus.
Acetylation of Sp1 by p300 regulates Sp1-c-Jun interaction
Jan-Jong Hung
Department of Pharmacology, College of Medicine, National Cheng Kung University
Acetylation of protein modulates the activities of nonhistone regulatory proteins and
plays a critical role in the regulation of cellular gene transcription. In this study, we
showed that the sp1 could be acetylated by p300 in vitro and in vivo. Acetylated sp1
increased the binding affinity with c-Jun at A431 cells. Furthermore, we try to
determine which residue(s) was acetylated by p300 by converted the lysine of sp1 to
alanine individually, and then study its role in the regulation of the gene transcription.
Our results describe a novel mechanism that sp1 acetylated by p300 increase binding
affinity with c-Jun to regulate the transcription activity of target gene.
NF-IL6 involved in the transcriptional regulation of EGF-induced COX-2 gene
expression
Ju-Ming Wang
Department of Pharmacology, College of Medicine, National Cheng Kung University
The CCAAT/enhancer binding protein  (C/EBP, CRP3, CELF, NF-IL6) regulates
gene expression and plays functional roles in many tissues, for example, in acute
phase response, adipocyte differentiation and mammary epithelial cell growth control.
In this study, we examined the expression of human C/EBP (NF-IL6) and COX-2
gene by epidermal growth factor (EGF) stimuli in A431 cell. The EGF inducibility
activity of the NF-IL6 promoter is abolished by mutating the sequence in the region
of -347/+9 bp containing the CRE or Sp1 sites. Both in vitro- and in vivo- DNA
binding assay showed the CREB binding activity was low in EGF-starved cells and
induced within 30min following EGF treatment of A431 cells; however the Sp1
binding activity was unchanged. Here we also provide the evidence of EGF-activated
p38MAPK/CREB signaling pathway regulates transcriptional activation of NF-IL6
gene. Furthermore, we found EGF-regulated COX-2 expression is mediated through
p38MAPK signaling pathway that corelated with NF-IL6 expression. By in vivo
DNA binding and siRNA assay, we demonstrated the EGF-induced NF-IL6 play a
functional role on COX-2 expression in A431 cells. Several studies showed that
sumoylation is important for gene inactivation. In our system, cotransfected Sumo-1
expression vector repressed EGF- or NF-IL6-induced COX-2 promoter activity. The
NF-IL6 can be sumoylated by in vivo- and in vitro- sumoylation assays; and the
sumoylated NF-IL6 loses the interaction ability of p300 by in vitro binding assay.
Using chromatin IP assay, we showed EGF stimulated the p300 recruitment and loss
of Sumo-1 modified proteins on COX-2 promoter region.
The gene regulation mechanism of thrombomodulin under epidermal growth
factor treatment in A431 cells
Joseph T. Tseng
Department of Pharmacology, College of Medicine, National Cheng Kung University
Thrombomodulin (THBD), recognized as an essential vessel wall cofactor of the
antithrombotic mechanism, is also expressed by a wide range of tumor cells. The
human epidermoid carcinoma cell line A431 expresses a high number of EGF
receptors and its growth is inhibited by nanomolar concentration of EGF. In this
report, we found that the mRNA expression of THBD was up-regulated under high
concentration of EGF treatment. Transcriptional and post-transcriptional regulation
was shown to involve in this phenomenon. EGF enhanced the mRNA stability of
THBD and the half-lives increased approx. 2-fold. The PI3 kinase inhibitor,
LY294002, rapidly destabilized TM mRNA. It indicated the PI3-kinase signaling
pathway involved in maintaining the TM mRNA stability. Transfections with a variety
of THBD 3’-UTR constructs identified multiple cis-acting AU-rich elements
responsible for mRNA stability.
Histone deacetylase inhibitors downregulate the expression of 12(S)-lipoxygenase
induced by epidermal growth factor
Ching-Jiunn Chen
Department of Pharmacology, College of Medicine, National Cheng Kung University
In studying the signal transduction of 12(S)-lipoxygenase expression by epidermal
growth factor in human epidermoid carcinoma A431 cells, we reported that
EGF-induced expression of 12(S)-lipoxygenase in A431 cells was mediated through
the Ras-ERK1/2 (p44/42 MAP kinase) and Ras-Rac-JNK signal pathways, and
subsequent induction of c-Jun led by ERK and JNK activation was essential for this
EGF response. To study the relationship between the activation of 12(S)-lipoxygenase
transcription and histone acetylation, trichostatin A (TSA) and sodium butyrate (SB)
were used. The HDAC inhibitors (TSA and SB) could enhance the acetylation level
of histone H4, but inhibit EGF-induced 12(S)-lipoxygenase enzyme activity and
protein expression. Interestingly, both inhibitors could enhance the reporter gene
expression of 12(S)-lipoxygenase promoter (-224~-7) induced by EGF. We analyzed
the mechanism of these inhibitors inhibiting 12(S)-lipoxygenase enzyme activity and
protein expression in three ways. First, whether these inhibitors affect on the signal
transduction of EGF-induced expression of 12(S)-lipoxygenase. Second, whether
these inhibitors can induce X factor and affect the stability of 12(S)-lipoxygenase.
Finally, whether these inhibitors can induce the other transcription repressors and
affect the promoter activity of 12(S)-lipoxygenase.
Molecular mechanism of interaction between vitamin D receptor and Sp1 in gene
regulation
Wen-Chun Hung
Institute of Biomedical Sciences, National Sun Yat-Sen University
Our recent study has demonstrated that vitamin D may stimulate p27Kip1 gene
expression via the Sp1 transcription factor binding site in the promoter. We find that
vitamin D treatment enhances the interaction between vitamin D receptor (VDR) and
Sp1 transcription factor and activates p27Kip1 expression via the VDR/Sp1 complex.
Co-immunprecipitation and GST-pull down assays indeed demonstrate that VDR and
Sp1 may physically interact with each other in vitro and in cells. By using
biotin-labeled double strand DNA probe, we find that the VDR/Sp1 complex may
bind to the oligonucleotide probe contained only Sp1 binding sites and the binding
activity of this complex is enhanced by vitamin D3. In addition, our results indicate
that vitamin D treatment induces de-phosphorylation of VDR. Our study provides
new insight of a novel molecular mechanism by which steroid hormones control the
expression of downstream target genes.
Significance of the amino terminus of Sp1 in gene regulation
Shwu-Jen Tzeng and Jin-Ding Huang
Department of Pharmacology, College of Medicine, National Cheng Kung University
Sp1 is a ubiquitous transcription factor that regulates a variety of mammalian genes.
Previous studies utilized a truncated Sp1 cDNA lacking the first 82 amino acids of the
Sp1 coding sequence (696 amino acids). It has been reported that transcriptional
activation of the rat CYP2D5 gene involves synergism between Sp1 and C/EBP.
Using Drosophila cells that lack endogenous Sp1 activity, we showed that Sp1 (696)
was more potent than Sp1 (778) in transactivation of CYP2D5 promoter. Similar
result was observed in rat Mrp3 promoter.
However, the amino terminus of Sp1
significantly interfered synergistic effect between Sp1 and C/EBP in CYP2D5
promoter, but moderately affect the synergistic effect between Sp1 and C/EBP in
Mrp3 promoter. The amino terminus of Sp1 prevent Sp1, C/EBP, or CBP/p300
synergistic transactivation of CYP2D5 promoter. We further construct truncated Sp1
cDNA lacking the first 28; 46; 64 amino acids of the Sp1 coding sequence (Sp1-N1;
Sp1-N2; Sp1-N3 respectively). We observed that these deletion constructs were
almost equal to Sp1 (696) in transactivation of CYP2D5 promoter in Drosophila SL2
cells. By using SUMOplot prediction software, it contains one SUMO targeted
motifs with high probability within the first 28 a.a. Mutational analyses of this site
enhance the activity of the CYP2D5 promoter about two to three folds, but the activity
of Mrp3 promoter was no obvious increase. Coexpression of Sp1 mutants with
C/EBP enhance the activity of Mrp3 promoter about two to three folds than Sp1
(778) with C/EBP. We found that Sp1 mutants with higher protein expression and
equal DNA binding in Western blotting and DAPA assay, and the ability of these
mutants translocation into nuclear is not altered. Taken together, it suggests that
sumoylation at the amino terminus of Sp1 regulated its transcriptional activity.
The mechanism for nuclear localization of MSP/RON receptor in human bladder
cancer
Pei-Yin Hsu1, Nan-Haw Chow2, and Hsiao-Sheng Liu3
1
Institute of Basic Medical Sciences, 2 Department of Pathology, 3 Department of
Microbiology & Immunology, College of Medicine, Nation Cheng Kung University
RON is a distinct receptor tyrosine kinase, belonging to the c-met receptor family. Our
recent cohort study revealed that RON-associated signaling is important in the
progression of human bladder cancer. In addition, MSP-induced phosphorylation of
RON enhances the migration, proliferation and anti-apoptosis of bladder cancer cells.
Intriguingly, both wild-typed and truncated form of RON translocated into the nucleus.
This study thus was performed to explore the mechanisms for nuclear
translocalization of RON in bladder carcinogenesis. We demonstrated that nuclear
RON is a full-length receptor, in de-phosphorylated form, and is MSP-independent.
The RON receptor collaborates with EGFR and importin 1 in translocation into the
nucleus. To our surprise, nuclear RON seems to co-localize with de-phosphorylated
EGFR. Phosphorylation of EGFR was observed after MSP treatment, in which RON
dissociates from the EGFR. Take together, the results of this investigation suggest a
cross-talk between membrane receptors in nuclear translocalization of RON. Further
investigation is underway to analyze the significance of EGFR phosphorylation
implicated in the observed RON-related biological functions and bladder
carcinogenesis.
p97Eps8 overexpression promotes colorectal cancer formation
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) was
overexpressed in v-Src-transformed cells; (ii) ectopically expressed p97Eps8 in
C3H10T1/2 fibroblasts promotes both in vitro and in vivo cellular transformation; and
(iii) ablation of Eps8 expression in v-Src-transformed cells (IV5) inhibits their
proliferation.
Thus, p97Eps8 may play an important role in Src-mediated
transformation. Indeed, when the amount of p97Eps8 was specifically reduced by
siRNA (small interference RNA) technology in IV5, its proliferation was also
significantly inhibited both in vitro and in vivo. Furthermore, analysis of several
cancer cell lines and human colorectal cancer specimens revealed the overexpression
of p97Eps8. Interestingly, SW620 cells with higher p97Eps8 expression than SW480
cells did exhibit increased growth rate. Reduced proliferation and tumor growth in
nude mice was observed in SW620 cells bearing eps8-siRNA. Thus, our results
have strongly indicated that p97Eps8, as an oncoprotein, participated in the formation
of human cancer and could be a potential target of anticancer drugs.
Proteomic and genetic expression profiles in the malignant pleural effusion
associated lung adenocarcinoma
Wu-Chou Su and Ming-Der Lai
Department of Internal Medicine and Biochemistry, College of Medicine, National
Cheng Kung University
To understand the mechanisms underlie the pathobiology and to identify biomarkers
for better diagnosis of malignant pleural effusion associated lung adenocarcinoma.
We collected MPEs from patients with lung adenocarcinomas for study. Proteins
precipitated from MPE were separated by 2D-PAGE and interested spots were
identified by MALDI-MS (Matrix-assisted Laser Desorption/Ionization-Mass
Spectrometry). RNA specimens extracted from cancer cells in MPE were subjected to
Affymetrix oligonucleotide microarray analysis. In proteomic studies, by comparing
protein expression patterns of smoking male with non-smoking female lung cancer
patients, we have identified three proteins overexpressed in female, non-smoking
group. They were α-1 inhibitor III precursor, apolipoprotein E, coatomer protein
complex, subunit β (COPB). The expression of these three proteins was low or
undetected in pleural effusion pooled from 10 patients with pneumonia. The
expression of COPB and APOE in malignant pleural fluids was confirmed by Western
blotting analysis and expression of APOE was found in cytoplasm of lung cancer cells
by immunohistochemistry. Fourteen specimens of cancer cells in MPE, three of
normal lung epithelial cell line (Bes2B), and ten normal part of lung tissue were
available for RNA isolation and the extracted RNAs were sent for Affymetrix
oligonucleotide microarray analyses. Using non-supervised, hierarchical clustering
analysis, normal lung tissue, lung adenocarcinoma cells from non-smoking females,
and lung adenocarcinoma from smoking males were separated into three groups
according to the genetic expression pattern. By using the statistical method-TNoM
(Threshold number of misclassification), we disclosed a 29-gene panel which
differentially expressed in non-smoking female and smoking male. The higher
expression of COPB and APOE was found both in proteomic and microarray studies,
suggesting a correlation exists between mRNA and protein expression. Since APOE
has been implicated in the invasiveness of human cancers, the findings from the study
are not only markers for diagnosis but also could be pathobiological factors.
Therefore, studying cancer cells and proteins in pleural effusion by proteomic and
microarray approaches is feasible.
ER stress, aberrant expression of cyclin A, genomic instability and nodular
hyperplasia of hepatocytes induced by hepatitis B virus pre-S mutant proteins:
implication in HBV-related hepatocarcinogenesis
Ih-Jen Su1, Hui-Ching Wang1, Ming-Derg Lai2, and Wenya Huang3
1
Division of Clinical Research, National Health Research Institutes, 2Department of
Biochemistry, and 3Department of Medical Laboratory Science and Biotechnology,
College of Medicine, National Cheng Kung University,
During chronic hepatitis B virus infection, the pre-S mutations have been linked to
hepatocellular carcinoma, especially the pre-S2 mutants. A specific pre-S2 mutant was
previously identified from marginal typed ground glass hepatocytes which might
confer growth advantages during hepatocarcinogenesis. We recently demonstrated
that this specific pre-S2 mutant protein could activate gene expression of cyclin A and
PCNA. An elevated expression of cyclin A, as judged by immunostaining, was also
evident on both ground glass hepatocytes and hepatoma tissues, suggesting a potential
role of pre-S2 mutant protein on cell cycle regulation. On HuH-7 cells, the expression
of pre-S2 mutant protein could enhance cell proliferation, as analyzed by
BrdU-incorporation assay. This mutant can induce abnormal multinucleated
phenotype, nuclear dysplastic changes and genomic instability on the pre-S2 mutant
transgenic mice. The induction of multinucleation and nodular hyperplasia on
transgenic mice may be associated with aberrant expression of cyclin A and ER stress
signaling pathways induced by the HBV pre-S2 mutant proteins. The hepatocyte
harboring this pre-S mutant may therefore represent a pre-neoplastic condition in the
process of HBV-related hepatocarcinogenesis.
Endoplasmic reticulum stress stimulates the expression of cyclooxygenase-2
through activation of NF-B and pp38 mitogen-activated protein kinase
Ming-Derg Lai
Department of Biochemistry and Molecular Biology, College of Medicine, National
Cheng Kung University
Expression of mutant proteins or viral infection may interfere with proper protein
folding activity in the endoplasmic reticulum (ER). Several pathways that maintain
cellular homeostasis are activated in response to these ER disturbances. In this report,
we investigated which of these ER stress-activated pathways induce COX-2, and
potentially oncogenesis. Tunicamycin and brefeldin A, two ER stress inducers,
increased the expression of COX-2 in ML-1 or MCF-7 cells. Nuclear translocation of
NF-B and activation of pp38 MAP kinase was observed during ER stress.
IB-kinase inhibitor Bay 11-7082 or IB kinase dominant negative mutant
significantly inhibited the induction of COX-2. PP38 MAP kinase inhibitor
SB-203580 or eIF2 phosphorylation inhibitor 2-aminopurine attenuated the nuclear
NF-B DNA binding activity and COX-2 induction. Expression of mutant hepatitis B
virus (HBV) large surface proteins, inducers of ER stress, enhanced the expression of
COX-2 in ML-1 and Huh-7 cells. Transgenic mice showed higher expression of
COX-2 protein in liver and kidney tissue expressing mutant HBV large surface
protein in vivo. Similarly, increased expression of COX-2 mRNA was observed in
human hepatocellular carcinoma tissue expressing mutant HBV large surface proteins.
In ML-1 cells expressing mutant HBV large surface protein, anchorage-independent
growth was enhanced, and the enhancement was abolished by the addition of specific
COX-2 inhibitors. Thus, ER stress due either to expression of viral surface proteins or
drugs can stimulate the expression of COX-2 through the NF-B and pp38 kinase
pathways. Our results provide important insights into cellular carcinogenesis
associated with latent endoplasmic reticulum stress.
Novel signaling to suppress the apoptosis in T cells
Bei-Chang Yang
Institute of Basic Medical Sciences and Department of Microbiology and
Immunology, College of Medicine, National Cheng Kung University
Although Fas/Fas-L signaling system is a prominent apoptosis-mediating pathway, it
induces gene expression such as IL-10 in several cells without leading to cell death.
We also observed that most of tumor-infiltrating cells isolated from FasL+ gastric
carcinoma were not apoptotic despite of caspase 3 activation. Two different studies
were carried out to elucidate the suppression of Fas-associated apoptosis. First, we
found that direct contact with tumor cells reduced the apoptosis in T cells. When
Jurkat cells contacted with tumor cells including glioma, hepatoma, or lung carcinoma,
they were less susceptible to the CH-11-induced apoptosis as revealed by decrease in
activation of caspase-8, -3 and -9, amelioration of loss of mitochondria membrane
potential, and reduction in breakage of DNA as compared to Jurkat cells alone in
culture. No new protein synthesis was required for the protective effect by cell-cell
contact for Jurkat cells. JUN and P38 MAPK kinases were not involved in this
protection. Proteomic study revealed that some new proteins were enhanced in Jurkat
cells in contact with glioma. Second, Fas signaling intrinsically activated p38 MAPK
pathway by it self to suppress caspase-cascade, which in turn reduced cell death. Fas
cross-linking by low dose CH-11 (1 ng/ml) stimulated mild phosphorylation of p38
MAPK in 12 h. Down regulation of FADD by siRNA or inactivation of caspase 8 by
Z-IETD eliminated the phosphorylation of p38MAPK in response to Fas treatment.
Suppression of p38 MAPK activity significantly enhanced the Fas-mediated apoptosis.
Blocking Fas receptor by antagonistic antibody ZB4 or trimmed the Fas signaling by
Z-IETD to inhibit caspase 8 activity totally eliminated the activation of caspase 3
even in the presence of p38 MAPK inhibitor indicating that the action of p38 MAPK
on caspase 3 was Fas signaling-specific. In summary, p38 MAPK pathway is an auto
feed-back switch in Fas signaling that can damp the progress of death event in T cells.
Moreover, tissue/tumor microenvironments also contribute to the immune responses
against tumor by regulating the viability of immune cells.
Mechanical property of collagen fiber-induced cell spreading and migration are
mediated by phosphorylation of ERK1/2 via lipid raft
Yu-Chih Hsu, Wen-Tai Chiu, Yang Kao-Wang and Ming-Jer Tang
Department of Physiology, College of Medicine, National Cheng Kung University
How mechanical impacts affect cell behaviors is a less studied issue. Here we
demonstrated that the low substratum rigidity of collagen gel triggered cell migration
in various cell lines. However, the mechanism of low rigidity-induced cell migration
has not been understood. In this study, MDCK cells cultured on collagen gel or
collagen gel-coated dish were employed to investigate how rigidity of collagen fiber
affected cell migration. We found that collagen gel induced phosphorylation of
ERK1/2 within 1 hour and the induction could last for more than 8 hours. Because
collagen gel coating did not alter ERK 1/2 activity, collagen gel-induced activation of
ERK1/2 is mediated by physical property, i.e. rigidity. The same phenomenon was
observed in HeLa and NIH3T3 cels. Inhibition of collagen-induced ERK1/2
phosphrylation by MEK inhibitors, UO126 and PD98059, resulted in reduction of cell
spreading as well as cell migration, as detected by time-lapse microscopic
examination. Interestingly, the collagen gel-induced ERK1/2 phosphorylation was
present in focal adhesion site, indicating the association of ERK1/2 activation with
cell spreading and migration. Moreover, filipin III, a cholesterol-binding factors,
completely alleviated the collagen gel-induced ERK1/2 phosphorylation. However,
siRNA of caveolin-1 did not block low rigidity-induced cell spreading. Taken together,
low rigidity of collagen fiber induces activation of ERK1/2, which may facilitate cell
spreading and migration. Lipid raft may play roles in mediating substratum
rigidity-induced ERK1/2 activation.
Substratum rigidity controls 1 integrin activation and FAK phosphorylation
Wei-Chun Wei, Yang-Kao Wang and Ming-Jer Tang
Department of Physiology, College of Medicine, National Cheng Kung University
Physical environment has been considered as an important factor in regulating cellular
behavior. We had shown that collagen gel, as a soft substratum, down-regulated the
level of focal adhesion complex proteins. Here we showed 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. Low
rigidity-induced decrease in FAK397 phosphorylation could be observed in various cell
lines including fibroblasts and transformed cells. We also checked the level of 1
integrin activation which is the upstream signal of FAK. We found that 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 or FAK397 phosphorlyation 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. We found that
disruption of actin cytoskeleton by cytochalasin D blocked FAK397 phosphorylation
but not 1 integrin activation while disruption of microtubules by colcemide had no
effect. Furthermore, we examined whether the membrane microdomain provided
signal leading to 1 integrin activation. 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.
Deregulation of AP-1 protein family in collagen gel –induced apoptosis mediated
by low substratum rigidity
Yao-Hsein Wang, Wen-Tai Chiu, Ching-Chou Wu, Hsien-Chang Chang and Ming-Jer
Tang
Department of Physiology and Institute of Basic Medical Sciences, College of
Medicine, and Institute of Biomedical Engineering, College of Engineering, National
Cheng Kung University
Previous studies in our laboratory demonstrated that collagen gel induced apoptosis in
epithelial cells via physical property of the gel, i.e. rigidity. In order to elucidate the
impact of the rigidity on cell life and death, we employed rheometer and dynamic
mechanical analyzer (DMA) to assess the Young’s module of collagen gel extracted
from rat-tails of different ages. Our results indicated that the levels of collagen
gel-induced apoptosis were inversely proportional to the rigidity of collagen gel.
Morphologically, LLC-PK1 cells grown on collagen gel exhibited more contracted
cell shape and pulled collagen fibers before they underwent apoptosis. Confocal
microscopic studies showed that collagen gel resulted in disruption of cytoskeleton,
thus internal organelles such as ER and mitochondria were restricted. To explore the
signal pathways involved in low rigidity-induced apoptosis, we analyzed changes of
ER proteins and caspases in cells cultured on collagen gel and collagen gel coated
dish. Collagen gel induced time dependent cleavage of calreticulin starting within 1
hour, which could be the initial trigger of collagen gel-induced apoptosis. Collagen
gel triggered marked activation of caspase-3 within 18 hour when apoptosis was
actively present. DEVD-fmk, an inhibitor for caspase-3, could not block collagen
gel-induced apoptosis. However, z-VAD-fmk, a pan-caspase inhibitor, or proteinase
inhibitor cocktail alone could prevent calreticulin degradation and inhibit caspase-3
activation, but did not completely block collagen gel-induced apoptosis. To
investigate whether collagen gel conferred stress signals to cultured cells through
activation of stress-activated protein kinase (SAPK/JNK), we assessed the MAP
kinase pathways. We found that MKK4/7, JNK and ERK were phosphorylated within
4 hour upon collagen gel. More interestingly, collagen gel induced rapid
down-regulation of c-Jun in the nucleus, a downstream signal protein of JNK, within
1 hour and triggered aberrant expression of c-fos in the cytosol, which lasted for at
least 24 h. Because collagen gel-induced up-regulation of c-fos could be prevented by
SP600125, but not UO126, it is likely that activation of JNK is the cause of aberrant
expression of c-fos. Although the cause-effect relationship between AP-1 deregulation
and apoptosis has not been established, our results suggest that activation of proteases
and de-regulation of AP-1 proteins might be involved in collagen gel-induced
apoptosis.
Molecular mechanism of ceramide-induced apoptosis: anti-apoptotic role of
lithium
Yee-Shin Lin
Department of Microbiology and Immunology, College of Medicine, National Cheng
Kung University
Ceramide, a second messenger of intracellular signaling, involves in multiple
physiological regulation including cell survival and apoptosis. Our study showed that
sequential activation of caspase-2 and caspase-8 is essential for ceramide-induced
mitochondrial apoptosis. Bcl-2 may rescue ceramide-induced mitochondrial apoptosis
through blockage on caspase-2 activation. Protein phosphatase 2A (PP2A)-mediated
Bcl-2 dephosphorylation is involved in caspase-2 activation induced by ceramide.
Interestingly, lithium promotes cell survival by inhibiting PP2A activity and caspase-2
activation. Furthermore, the requirement of GSK-3 in ceramide-induced
mitochondrial apoptosis was demonstrated. The involvement of PP2A and PI3K/PKB
in GSK-3-mediated apoptotic signaling was shown, which was also inhibited by
lithium treatment. Further studies using microarray analysis showed that
ceramide-mediated upregulation of thioredoxin interacting protein (TXNIP) might be
involved in apoptotic pathway via a transcription-dependent mechanism. Lithium
causes a negative regulatory effect against ceramide-upregulated TXNIP expression.
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.