From the Departments of ¶¶ Medicine,
Cellular and Developmental Biology, and
Pathology,
Vanderbilt University Medical Center, Nashville, Tennessee 37232, the
§ Dana-Farber Cancer Institute and Department of Cell
Biology, Harvard Medical School, Boston, Massachusetts 02115, ¶ Psychiatric Genomics, Inc., Gaithersburg, Maryland 20878, ** Cutaneous Biology Research Center, Massachusetts General
Hospital and Harvard Medical School, Charlestown, Massachusetts 02129, and §§ Nuclear Receptor Discovery Research,
GlaxoSmithKline, Research Triangle Park, North Carolina 27709
Received for publication, August 7, 2002, and in revised form, November 18, 2002
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ABSTRACT |
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Peroxisome proliferator-activated
receptor The pathways that induce intestinal epithelial
differentiation are complex and multigenic (1, 2). Two biologically
important regulators of intestinal epithelial cell growth are the
peroxisomal proliferator-activated receptor- The TGF- Thus, although both PPAR Cell Culture and Materials
The MOSER S (M-S) (21) and MOSER R (M-R) (22) colon
carcinoma lines were a gift of M. Brattain (University of Texas Health Sciences, San Antonio, TX). The FET cell line was a gift of W. Grady
(Vanderbilt University, Nashville, TN) and the CBS cell line was
a gift of H. Moses (Vanderbilt University). COS7 cells were purchased
from ATCC (Rockville, MD). Cells were grown in Dulbecco's modified
Eagle's medium (Invitrogen) supplemented with 10% fetal
bovine serum (Hyclone), L-glutamine (2 mmol/liter), penicillin (100 units/ml), and streptomycin (100 µg/ml) in a 5% CO2 atmosphere with constant humidity. TGF- High Density cDNA Microarray Screening
Human named genes GENEFILTERS Release I blots (Research Genetics
Inc., Huntsville, AL) were probed with [33P]dCTP-labeled
reverse transcriptase-derived cDNA. The cDNA was synthesized
from RNA isolated from exponentially growing M-S cells that had been
exposed for 12 or 24 h to one of the following treatments: 0.1% 4 mM HCl containing 0.1% bovine serum albumin, 0.1%
Me2SO, 1 µM rosiglitazone, or 2 ng/ml
TGF- Northern Hybridization Analysis
Northern blot analysis was performed as described
previously (23). Although representative Northern blot images are
shown, all Northern blot experiments were repeated a minimum of two
times. Briefly, total RNA (20 µg) was fractionated on a 1.2%
agarose-formaldehyde gel and transferred to a Hybond-NX nylon membrane
(Amersham Biosciences). Filters were prehybridized for 4 h
at 42 °C in Ultrahyb (Ambion, Austin, TX). Hybridization was
conducted in the same buffer in the presence of 32P
radiolabeled cDNA of a partial fragment of human TSC-22. Blots were
washed 4× for 15 min at 50 °C in 2× SSC, 0.1% SDS and once for 30 min in 1× SSC, 0.1% SDS. Membranes were then exposed to a
PhosphorImager screen and images were analyzed using a Cyclone Storage Phosphor system. Quantification of mRNA intensities for Fig. 4 (A-D) was done using Optiquant Software
(Hewlett-Packard).
Western Blot Analysis
Exponentially growing cells were harvested in ice-cold 1×
phosphate-buffered saline and cell pellets were lysed in RIPA buffer. Centrifuged lysates (50 µg) from each cell line were fractionated on
a 4-20% gradient SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane (PerkinElmer Life
Sciences). Membranes were blocked for 1 h at room
temperature in Tris-buffered saline containing 0.1% Tween 20 (TBST)
and 5% powdered milk. The primary antibody was then added and
incubated at room temperature for 2 h or overnight at 4 °C. The
following primary antibodies were used: monoclonal anti-hemagglutinin
(HA) antibody clone HA.11 (1:1000; Babco, Richmond, CA), monoclonal anti-FLAG antibody clone M2 (1:500; Sigma), monoclonal anti-keratin 20 antibody clone Ks20.8 (1:500; NeoMarkers, Fremont, CA), goat polyclonal
anti-p21 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit
polyclonal anti-TSC-22
antibody2 (1:100). This was
followed by incubation with the appropriate horseradish
peroxidase-conjugated secondary antibody (Jackson Labs) at a dilution
of 1:50,000 for 1 h. Detection of immunoreactive polypeptides was
accomplished using an enhanced chemiluminescence system (Amersham Biosciences).
DNA Constructs
For all PCR reactions, Pfu Turbo Taq Polymerase
(Stratagene, La Jolla, CA) was used. Every construct generated was
sequenced to ensure the absence of any unwanted mutations. Full-length
TSC-22 was cloned using PCR from a cDNA library of M-S
cells treated with 1 µM rosiglitazone for 24 h. The
product was cloned into the pPCR-Script cloning vector (Stratagene, La
Jolla, CA) and this plasmid was utilized as a template for all
subsequent PCR reactions. For all primers, the 5' primer was designed
to include a NotI site and nucleotides encoding the
full-length HA or FLAG epitope. Each 3' primer was designed to include
a BglII site. For HA TSC-22 wild-type (wt) or FLAG TSC-22
wt, the 5' primer included a partial region of TSC-22
starting at the second codon and the 3' primer included a partial
region starting at codon 145 (stop codon). For HA TSC-22dn, in which
both repressor domains (RD) were deleted, the 5' primer included a
partial region of TSC-22 beginning at codon 38. The 3'
primer included a stop codon and a partial region starting at codon
102. For HA TSC-22dncon, both RDs were deleted and two highly conserved
leucine residues in the leucine zipper domain were mutated to alanine
(L91A and L97A). The 5' primer was identical to that used for HA
TSC-22dn. The 3' primer was also the same except for a mismatch at
codons 91 and 97 such that the amplified product would contain the L91A and L97A mutations. See Fig. 5A for a schematic of all
constructs generated. Each PCR product was digested with
NotI/BglII and subcloned into the pcDNA3.1Zeo
expression vector (Invitrogen, La Jolla, CA).
Transient Transfections
Exponentially growing COS7 cells were transiently transfected
with the appropriate plasmid using FuGENE 6 (Roche Molecular Biochemicals, Indianapolis, IN) at a lipid:DNA ratio of 3.5:1 and with
a DNA concentration of 1.5 µg/ml in Opti-MEM (Invitrogen). 6 h
post-transfection, regular media was added back and the cells were
allowed to grow for another 48 h prior to harvest.
Immunoprecipitations
Anti-HA affinity matrix (100 µl; Roche Molecular Biosciences)
was added to 500 µg of COS7 cell lysate and the cells were incubated in NET-N buffer (150 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl, pH 8.0, 0.5% Nonidet P-40, and 10% glycerol)
at 4 °C for 4 h. Beads were then washed 3× with NET-N buffer.
Protein was eluted by the addition of 1× SDS loading buffer followed
by a 5-min incubation at 100 °C.
Stable Cell Lines
Four different vectors were utilized to generate eight unique
pools of stable transfected cells: 1) pcDNA3.1Zeo (pools designated as M-S VECTOR clones A and B); 2) HA TSC-22wt/pcDNA3.1Zeo (M-S TSC
WT clones A and B); 3) HA TSC-22dn/pCDNA3.1Zeo (M-S TSC DN clones A
and B); and 4) HA TSC-22dncon/pcDNA3.1Zeo (M-S TSC DNCON clones A
and B).
Transfection--
M-S cells were transfected with the
appropriate vector using FuGENE 6 at a lipid:DNA ratio of 3:1 and a DNA
concentration of 5 µg/ml in Opti-MEM. After 6 h of transfection,
normal media was added back and the cells were allowed to grow for
another 72 h.
Selection--
Cells were then split into media containing 200 µg/ml zeocin (Invitrogen, La Jolla, CA) and selected for 2 weeks.
Resistant clones were combined into two unique pools and expanded.
Zeocin was maintained in the media prior to all experiments at a dose of 100 µg/ml.
Cell Growth Measurements
Cells were plated at a density of 5 × 104 and
replaced the next day with Dulbecco's modified Eagle's medium
containing 10% charcoal-stripped fetal bovine serum and rosiglitazone
or TGF- In Situ Hybridization
In situ hybridization was performed as described
previously (24). Sense or antisense 35S-labeled cRNA probes
were generated from human TSC-22. The probes had specific activities at
2 × 109 disintegrations per minute (dpm)/µg.
Sections hybridized with the sense probes did not exhibit any positive
autoradiographic signals and served as negative controls.
Cellular Response of Colorectal Cancer Cells to PPAR Identification of TSC-22 as a PPAR Transcriptional Regulation of TSC-22 by PPAR
As TSC-22 has not previously been shown to be a target of PPARs, we
wanted to determine the specificity and selectivity of this induction.
Cells were treated with two structurally distinct PPAR The Ability of PPAR Overexpression of Wild-type TSC-22 Inhibits Colon Epithelial Cell
Growth and Induces Elevated Levels of p21--
The functional role of
TSC-22 in mediating any of the biological effects induced by either
PPAR
Each of these three constructs (plus empty vector) was used to generate
eight unique pools of stably transfected cells. Each of the pooled cell
lines was found to express relatively equivalent protein levels of the
integrated cDNA (Fig. 8, C and D). The two different pools of M-S cells expressing TSC-22 wt were found to have
significantly reduced growth rates compared with vector-transfected cells (Fig. 9A). They also
displayed higher levels of p21 protein but showed no difference in the
levels of keratin 20 protein (Fig. 9B).
Overexpression of Dominant Negative TSC-22 Partially Inhibits
PPAR The complex mechanisms by which the undifferentiated stem cells of
the intestine give rise to differentiated cells with specialized functions remain incompletely understood. Some of the key pathways that
govern this process in the intestine have been identified from
understanding genetic lesions found in colorectal cancer (36). For
example, a majority of human colorectal cancers contain a loss of
function mutation in the tumor suppressor gene adenomatous polyposis
coli (37). Subsequent studies have suggested a critical role for
this gene in exerting strict growth controls such that the renewing
population of intestinal epithelial cells is kept at a constant number.
Likewise, loss of function mutations have been identified in genes
involved in the TGF- There are several reasons why the results presented here are likely to
be biologically relevant. First of all, TSC-22 is localized to the most
differentiated epithelia in the normal human colon. PPAR TSC-22 is highly conserved during evolution, with the human protein
sequence being 98.5% identical to the mouse and rat proteins (40).
TSC-22 has been shown to a have a solution structure similar to members
of the bZIP family of transcription factors (41). Similar to bZIP
proteins, TSC-22 contains a leucine zipper domain and, analogous to the
basic domain in bZIP family members, has a highly conserved sequence
known as the TSC domain or box. In addition, TSC-22 is also homologous
to the Drosophila melanogaster gene shortsighted
(shs) or bunched, which is required for normal development
of oocytes, larval peripheral nervous system, and adult eye and wing
(42-44). Recent evidence also implicates an important role for TSC-22
in the developing feather tract (45). There is also evidence suggesting
that TSC-22 can negatively regulate the growth of cancer cells. For
example, antisense expression of TSC-22 in a salivary gland carcinoma
line inhibits the growth of these cells in vivo (46) and
overexpression of TSC-22 appears to sensitize carcinoma cells to
certain anti-cancer chemotherapeutic drugs (47).
Evidence to date suggests that TSC-22 is part of a family of proteins
that act as transcriptional repressors. As mentioned earlier, TSC-22
has been shown to homodimerize and exhibit transcriptional repressor
activity when fused to a heterologous DNA-binding domain (35). In fact,
our results with the TSC-22dn protein in which both repressor domains
were deleted provide functional evidence in a biological system for the
importance of these domains. This notion is further supported by the
finding that the TSC-22 Drosophila homolog,
bunched, is a powerful repressor of the enhancer trap reporter A359 (44). It is also possible that TSC-22 may modify gene
expression through protein-protein interactions. Recently, a TSC-22
homologue (termed THG-1) was cloned and found capable of forming
heterodimers with TSC-22 (35). (THG-1 was not found to be expressed in
the M-S cells.) Exactly how either a TSC-22 homodimer complex or a
TSC-22·THG-1 heterodimer complex can regulate the expression
of a particular target gene is unknown.
One possible target gene of TSC-22 may be p21. However, at this point
it is unclear whether TSC-22 increases p21 levels directly or
indirectly. Although our work provides evidence that TSC-22 is involved
in the signaling pathway by which both PPAR In summary, the data reported here point to a previously unidentified
role for the putative transcriptional repressor TSC-22 as a regulator
of intestinal epithelial cell differentiation. A large percentage of
advanced colorectal tumors lose their responsiveness to growth
inhibition induced by TGF- (PPAR
) and transforming growth factor-
(TGF-
) are
key regulators of epithelial cell biology. However, the molecular
mechanisms by which either pathway induces growth inhibition and
differentiation are incompletely understood. We have identified
transforming growth factor-simulated clone-22
(TSC-22) as a target gene of both pathways in
intestinal epithelial cells. TSC-22 is member of a family of leucine
zipper containing transcription factors with repressor activity.
Although little is known regarding its function in mammals, the
Drosophila homolog of TSC-22,
bunched, plays an essential role in fly development. The
ability of PPAR
to induce TSC-22 was not dependent on an intact
TGF-
1 signaling pathway and was specific for the
isoform. Localization studies revealed that TSC-22 mRNA is enriched in the
postmitotic epithelial compartment of the normal human colon. Cells
transfected with wild-type TSC-22 exhibited reduced growth rates and
increased levels of p21 compared with vector-transfected cells.
Furthermore, transfection with a dominant negative TSC-22 in which both
repressor domains were deleted was able to reverse the p21 induction
and growth inhibition caused by activation of either the PPAR
or
TGF-
pathways. These results place TSC-22 as an important
downstream component of PPAR
and TGF-
signaling during
intestinal epithelial cell differentiation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(PPAR
)1 and transforming
growth factor-
(TGF-
) pathways. PPAR isoforms
,
, and
constitute a family of orphan nuclear hormone receptors that serve to
integrate dietary fat intake with changes in the expression of genes
involved in both fatty acid oxidation and storage (3). PPAR
was
originally defined as an essential component of the adipocyte
differentiation program (4). However, the range of biological functions
that PPAR
regulates is much wider than originally thought and the
receptor has been shown to modulate the growth of a wide variety of
cell types (5-7). This is perhaps most evident in the colon, where
activation of the receptor inhibits the growth of a broad range of
colon cancer cell lines and induces markers of differentiation (8, 9).
The effects of PPAR
on colorectal carcinogenesis may be dependent on
the model system used because activators of the receptor slightly
increase the number of colonic adenomas in the min mouse (a
murine model of familial adenomatous polyposis) (10, 11). However,
~8% of primary colorectal tumors were found to harbor a loss of
function mutation in the PPAR
gene emphasizing the putative role of
this receptor as a tumor suppressor in humans (12).
family of growth factors regulate a plethora of biological
processes including embryonic development, wound healing, angiogenesis,
proliferation, and differentiation of cells (reviewed in Ref. 13). This
latter function has been well defined in the colon, where TGF-
is a
potent inhibitor of colonic epithelial cell growth (14, 15). Loss of
normal TGF-
responsiveness occurs commonly during the development of
colorectal cancers associated with microsatellite instability and
genetic lesions that disrupt the TGF-
pathway have been identified,
including loss of function mutations in the TGF-
type II receptor
(16, 17), SMAD4 (18), and SMAD2 (19). Under these circumstances
continued expression of TGF-
paradoxically leads to enhanced tumor
growth through stimulation of angiogenesis, extracellular matrix
production, and immuno suppression (20).
and TGF-
are key regulators of
epithelial cell biology, the molecular mechanisms by which either pathway induces growth inhibition and differentiation are incompletely understood. Here we have identified transforming growth factor simulated clone-22 (TSC-22) as a target gene of both
pathways in intestinal epithelial cells. Functional studies with
wild-type and dominant negative forms of TSC-22 suggest that TSC-22 is
an important downstream component of PPAR
and TGF-
signaling
during intestinal epithelial cell differentiation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,
cycloheximide, and
5,6-dichloro-
-D-ribofuranosyl-benzimidazole (DRB) were
purchased from Sigma. Synthetic PPAR and RXR ligands were synthesized
at GlaxoSmithKline (Research Triangle Park, NC) and dissolved in Me2SO. RNA was isolated using the TRI reagent (Molecular
Research Center, Cincinnati, OH). For all experiments in which TGF-
1
or a PPAR ligand were added, the cells were grown in the above media except that regular fetal bovine serum was replaced by
charcoal-stripped fetal bovine serum (Hyclone).
1. Membranes were hybridized and washed according to the
manufacturer's instructions. The blots were imaged using the Cyclone
Storage Phosphor System (Hewlett-Packard) and imported into the
Pathways Software (Research Genetics Inc., Huntsville, AL) to identify
differences in the intensity of cDNA spots between different treatments.
1. Cells were counted at the indicated times using a Coulter
counter. Each experiment was done in triplicate.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
TGF-
1--
We noticed a striking similarity in the cellular
response to either TGF-
1 or PPAR
ligands in colorectal cancer
cells with intact PPAR
and TGF-
signaling pathways. For example,
in the M-S, CBS, and FET colon carcinoma lines, exposure to the high affinity PPAR
ligand rosiglitazone (25) or TGF-
1 results in accumulation of cells in the G1 phase of the cell cycle
(data not shown) and a decrease in cell growth (Fig.
1A). Moreover, in all three
cell lines, addition of either agent results in an increase in the p21
(Fig. 1B) and keratin 20 (data not shown). Elevated levels
of the cyclin-dependent kinase inhibitor p21 (26, 27) and
keratin 20 (28) are associated with intestinal epithelial differentiation in various model systems and p21 is a well
characterized TGF-
-regulated gene (29). Because both PPAR
and
TGF-
1 regulate such a wide spectrum of physiological processes, the
genomic response to either pathway is complex. This makes it difficult
to identify those target genes that play functionally important roles
in cell growth and differentiation. These data led us to hypothesize
that one way to identify relevant target genes of PPAR
and TGF-
1 in the setting of colon epithelial cell growth and differentiation is
to focus on the common subset of downstream target genes regulated by
both pathways.
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Fig. 1.
The PPAR ligand
rosiglitazone or TGF-
1 induces growth
inhibition and increases in protein levels of p21 in a panel of colon
epithelial cell lines. A, M-S, FET, or CBS cell
lines were treated with vehicle (0.1% Me2SO + 0.1% bovine
serum albumin), 1 µM rosiglitazone, or 2 ng/ml TGF-
1
and cells were counted at days 2, 4, and 6 post-treatment. Each
data point represents the mean of two independent
experiments, each done in triplicate. Error bars = S.E.
B, M-S, FET, and CBS cell lines were treated with
vehicle, 1 µM rosiglitazone, or 2 ng/ml TGF-
1
for 4 days after which total protein lysates were probed for levels of
p21 by immunoblot.
and TGF-
Target Gene in
Colon Epithelial Cells--
Microarray analysis was utilized to
determine the genomic response of one of these lines, the M-S cells,
after exposure to rosiglitazone or TGF-
1 for 12 or 24 h. A
subset of genes was commonly regulated by either pathway, including
several members of the keratin and carcinogenic embryonic antigen
superfamilies. However, time course experiments suggested that these
genes were not likely to be direct targets of PPAR
or TGF-
and
may represent end points (rather than effectors) of the differentiation
process. One promising candidate rapidly induced by either treatment
was TSC-22, a gene originally identified as a
TGF-
1-stimulated gene in osteoblast cells (30). Northern blot
analysis confirmed that either PPAR
or TGF-
could induce
TSC-22 in the M-S, CBS, and FET cell lines (Fig.
2). We further became interested in
studying this gene when in situ hybridization of TSC-22 in
the normal colon demonstrated that its expression was enriched in the
postmitotic epithelial compartment of the normal human colon, where the
most differentiated enterocytes reside (Fig.
3).
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Fig. 2.
TSC-22 is a downstream target of both
PPAR and TGF-
1 in
colon epithelial cells. M-S, FET, and CBS cell lines were treated
with vehicle, 1 µM rosiglitazone, or 2 ng/ml TGF-
1 for
12 h after which total RNA was collected and probed for TSC-22
levels by Northern blot (20 µg RNA/lane).
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Fig. 3.
TSC-22 is localized to the postmitotic
epithelial compartment of the normal human colon. The
surface of the human intestine is divided into an epithelial mucosal
layer and submucosal layer containing supportive connective tissue,
lymphatics, and vasculature. Within the mucosal layer, undifferentiated
stem cells located at the base of invaginated crypts give rise to cells
that migrate toward the lumen as they further differentiate into
specialized enterocytes. Sections of normal colon were probed with
antisense TSC-22 using in situ hybridization and a
representative section is shown.
and
TGF-
1--
The M-S cells were chosen as a system to study TSC-22
regulation by PPAR
or TGF-
1 and to test the hypothesis that
TSC-22 is a regulator of intestinal epithelial differentiation. The
induction of TSC-22 by either PPAR
or TGF-
1 was both
time- and dose-dependent (Fig.
4, A-D). Following treatment
with either rosiglitazone or TGF-
1, anti-TSC-22 antiserum detected
an appropriately sized polypeptide, which was absent in untreated cells
(Fig. 4E). We conclude that TSC-22 protein levels increase
as well. TSC-22 has been shown to be a direct target gene of TGF-
1
(30). We were interested in determining whether it is also a target for
PPAR
as well. Cells were pretreated with the protein synthesis
inhibitor cycloheximide or the RNA polymerase II inhibitor DRB followed by rosiglitazone. DRB co-treatment blocked the ability of PPAR
to
induce TSC-22 suggesting that the ability of PPAR
to increase steady-state RNA levels of the gene was dependent on de novo
transcription rather than through an increase in mRNA stability
(Fig. 5). Furthermore, whereas
cycloheximide slightly induced TSC-22, co-treatment of rosiglitazone
and cycloheximide led to a superinduction (Fig. 5), suggesting that
de novo protein synthesis was not required for PPAR
to
induce TSC-22. This result suggested that PPAR
was not
inducing TSC-22 by first increasing the levels of TGF-
and in fact
rosiglitazone was not able to induce TGF-
1 in the parental M-S cells
(data not shown).
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Fig. 4.
Time and dose-dependent induction
of TSC-22 by PPAR and
TGF-
1. A and
B, exponentially growing M-S cells were treated with 1 µM rosiglitazone at the indicated time points or
increasing doses of rosiglitazone (24 h) and cells were harvested for
RNA isolation. TSC-22 mRNA levels was detected by Northern blotting
(20 µg of total RNA/lane) and quantified by PhosphorImager analysis.
C and D, M-S cells were treated with 2 ng/ml
TGF-
1 or increasing doses of TGF-
1 (12 h) and TSC-22 mRNA
levels were detected by Northern blotting (20 µg of total RNA/lane)
and quantified by PhosphorImager analysis. E, M-S cells
were treated with 2 ng/ml TGF-
1 or 1 µM rosiglitazone
for 12 h and whole cell lysates were probed by immunoblot analysis
for TSC-22 using a polyclonal anti-TSC-22 antibody.
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Fig. 5.
TSC-22 is a direct target of PPAR .
MOSER cells were pretreated with 0.1% Me2SO
(DMSO), 10 µg/ml cycloheximide (CHX), or 25 µg/ml DRB for 30 min followed by treatment with 1 µM
BRL49653 for 3 (CHX) or 3 and 6 h (DRB).
TSC-22 mRNA levels was detected by Northern blotting (20 µg of
total RNA/lane).
ligands (the
thiazolidinedione-based agonist rosiglitazone or the tyrosine-based
agonist GW7845 (31)), an irreversible PPAR
antagonist (GW9662 (32)),
or rosiglitazone plus GW9662. TSC-22 was induced by both
PPAR
agonists and the induction by rosiglitazone could be blocked by
co-treatment with the PPAR
antagonist (Fig.
6). Cells were also treated with a
PPAR
selective ligand (GW7647 (33)), a dual PPAR
/
ligand
(GW2433 (34)), an RXR specific ligand (LG100268), or a combination of
rosiglitazone and LG100268. Despite the fact that M-S cells express
both PPAR
and PPAR
(data not shown), neither of these other two
PPAR isoforms was able to regulate TSC-22 expression (Fig. 6).
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Fig. 6.
TSC-22 is specifically and selectively
induced by PPAR . M-S cells were treated with the following
compounds for 24 h (dose and PPAR isoform selectivity in
parentheses): rosiglitazone (1 µM, PPAR
), GW7845 (1 µM, PPAR
), GW9662 (5 µM, PPAR
antagonist), Rosi + GW9662, GW7647 (1 µM, PPAR
),
GW2433 (1 µM, PPAR
and PPAR
), LG100268 (0.5 µM, RXR), and Rosi + LG100268. TSC-22 mRNA levels was
detected by Northern blotting (20 µg of total RNA/lane).
DMSO, Me2SO.
to Induce TSC-22 Is Independent of
TGF-
1--
To further clarify the issue of whether PPAR
signaling in the colon was dependent on the TGF-
pathway, the M-R
cell line was utilized. The M-R line is a subclone of the parental M-S
cells that are relatively refractory to the growth inhibitory effects of TGF-
(22). PPAR
, but not TGF-
1, could induce expression of
TSC-22 in the M-R cells (Fig.
7A). Finally, activators of
PPAR
inhibited the growth of either cell line by equivalent amounts, whereas the M-S-R cells (as has been previously reported) were relatively resistant to TGF-
induced growth inhibition (Fig. 7B).
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Fig. 7.
The induction of TSC-22 by
PPAR is not dependent on an intact
TGF-
1 signaling pathway.
A, M-R cells (a naturally identified TGF-
1 resistant
clone) were treated with 1 µM rosiglitazone or 2 ng/ml
TGF-
1 for 12 h. TSC-22 mRNA levels was detected by Northern
blotting (20 µg of total RNA/lane). B, M-S and M-R
cells were treated with vehicle (0.1% Me2SO + 0.1% bovine
serum albumin), 1 µM rosiglitazone, or TGF-
1 for 6 days after which time the number of viable cells were counted. Values
are expressed as percent of vehicle-treated cells. Each experiment was
performed in triplicate and the values represent the mean of two
independent experiments. Error bars = S.E.
or TGF-
is unknown. To address this issue we focused on two
experimental strategies: 1) to determine whether expression of TSC-22
in the M-S cells could recapitulate any of the phenotypic changes
induced by PPAR
or TGF-
1, and 2) to determine whether inhibition
of normal TSC-22 function blocks the phenotypic changes induced by
either treatment. Kester et al. (35) have defined 4 functional domains within TSC-22 (Fig. 8A). Using both glutathione
S-transferase pull-down and mammalian two-hybrid assays,
they demonstrated that TSC-22 could form homodimers via the leucine
zipper domain. They also showed that TSC-22 has transcriptional
repressor activity when fused to a heterologous DNA-binding domain and
identified two domains within TSC-22, RD 1 and 2, which were in
large part responsible for this effect. Finally, they were able to
demonstrate that titration of a mutant TSC-22 in which both RD 1 and 2 were deleted could inhibit the repressor activity of the wild-type
protein and thus act as a dominant-negative (dn) inhibitor. Based on
these results, we constructed three different HA-tagged TSC-22
constructs for stable introduction of this gene into the M-S cells:
TSC-22 wt, TSC-22dn (in which both RD 1 and 2 were deleted), and
TSC-22dncon (which, in addition to both RD domains being deleted,
contains mutations in two highly conserved leucine residues within the
leucine zipper domain) (Fig. 8A).
Co-immunoprecipitation experiments confirmed that TSC-22dn, but not
TSC-22dncon, could dimerize with TSC-22 wt (Fig. 8B). This
last construct was made to help properly interpret any potential artifacts because of expression of TSC-22dn that were independent of
its ability to dimerize and inhibit the function of the wild-type protein.
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Fig. 8.
Expression of full-length and mutant TSC-22
constructs. A, schematic of TSC-22 constructs
outlining the following domains: RD 1 and 2, the TSC box, and leucine
zipper (LZ domain). TSC-22 RD1/2 (TSC-22 DN), the RD 1 and
2 domains were deleted. TSC-22
RD1/2L91/97A (TSC-22 DNCON): the RD 1 and 2 domains were deleted and two highly conserved leucine residues in
the leucine zipper domain were mutated to alanine (L91A and L97A) to
disrupt its ability to dimerize with TSC-22 wt. B,
TSC-22dn, but not TSC-22dncon, can dimerize with TSC-22 wt. COS7 cells
were transiently transfected with the indicated plasmid, and lysates
were used for straight immunoblotting (50 µg of total protein/lane)
or immunoprecipitation (500 µg of total protein/lane) followed by
immunoblotting. C and D, stable expression
of TSC-22 wt (C) and TSC-22dn and TSC-22dncon (D)
in individual pooled clones of zeocin-resistant M-S cells. For both
gels, 50 µg of total protein was loaded per lane and probed with an
anti- HA antibody.
View larger version (26K):
[in a new window]
Fig. 9.
Wild-type TSC-22 inhibits cell growth and
leads to increased levels of p21, but not keratin 20. A, each pooled line was plated and viable cells were
counted at 2, 4, and 6 days. Each experiment was done in triplicate and
each data point represents the mean of two independent experiments.
Error bars = S.E. B, M-S vector clone A
and M-S TSC WT clone A cell lines were plated and protein lysates were
collected at days 1-4. 50 µg of total protein was loaded on each
lane and membranes were probed with anti-p21 or anti-keratin 20 antibodies.
Ligand and TGF-
1-induced Growth Inhibition and p21
Induction--
We next tested the ability of TSC-22dn and TSC-22dncon
to block the ability of PPAR
or TGF-
to inhibit the growth of M-S cells. In the two different pools of cells expressing TSC-22dn, the
ability of PPAR
or TGF-
1 to inhibit growth was reduced by ~60%
(Fig. 10A). Importantly, no
differences in the inhibitory activity of either PPAR
or TGF-
1
were seen when comparing vector and TSC-22dncon-transfected cells (Fig.
10A). Northern blot analysis confirmed that PPAR
and
TGF-
1 could still induce TSC-22 in these cell lines (data not
shown). Finally, the ability of rosiglitazone or TGF-
1 to induce
p21, but not keratin 20, was greatly diminished in M-S cells expressing
TSC-22dn but not TSC- 22dncon (Fig. 10B).
View larger version (24K):
[in a new window]
Fig. 10.
Dominant negative TSC-22 blocks the ability
of PPAR or TGF-
to
induce p21 and inhibit cell growth. A, each
indicated cell line was plated and the number of viable cells were
counted at day 6 following treatment with 1 µM
rosiglitazone or 2 ng/ml TGF-
1. Values are represented as % of
vehicle-treated cells and represent the mean from two independent
experiments. Error bars = S.E. B, each
indicated cell line was treated with vehicle, 1 µM
rosiglitazone, or 2 ng/ml TGF-
1, and protein lysates were collected
after 4 days. 50 µg of total protein was loaded and membranes were
probed with anti-p21 or anti-keratin 20 antibodies.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
signaling pathway and PPAR
. Further studies
have shown that activation of either of these pathways in cell culture
models can lead to growth inhibition and the induction of markers of
differentiation. These experiments emphasize the biological relevance
of these two pathways in intestinal epithelial cell biology. In this
study, we used microarray technology to identify TSC-22 as a
target gene of both PPAR
and TGF-
in intestinal epithelial cells.
We further show using wild-type and dominant negative forms of TSC-22
that this gene plays an important role in the ability of either PPAR
or TGF-
to inhibit cell growth.
and
TGF-
1 have been previously shown to be most strongly expressed
in vivo in the most differentiated epithelial cells of the
colonic mucosa (38, 39). Thus, all three of these genes appear to be
co-localized in the normal human colon to the most differentiated
enterocytes. Second, PPAR
and TGF-
1 were shown to regulate TSC-22
in multiple colon cancer cell lines, emphasizing the fact that the
present findings are not simply limited to one unique colon cancer cell
clone. In addition, the functional experiments in which a dominant
negative TSC-22 could reverse the growth inhibition caused by PPAR
ligands or TGF-
1 are likely because of its ability to inhibit normal
TSC-22 function. This is because no such reversal was seen with a
control dominant negative construct that cannot dimerize with and
inhibit wild-type TSC-22. We also provide evidence that there is
specificity to this putative signaling system. For example, TSC-22 was
not induced by PPAR
or -
(neither of which induces epithelial
cell differentiation). In addition, the induction of TSC-22 does not
appear to be a general phenomenon of agents that inhibit cell growth
because the RXR ligand LG100268 did not induce TSC-22 (Fig. 6) but does
inhibit the growth of multiple cell types, including the M-S cells
(data not shown). Finally, TSC-22 did not have any effect on the
expression of keratin 20 suggesting that TSC-22 is involved in one
facet of the differentiation program (control of cell growth) but is
not involved in pathways that lead to changes in the expression of
certain structural proteins.
and TGF-
induce p21,
there are other mechanisms by which activation of either pathway may
modulate p21 expression. For example, TGF-
has been shown to
activate p21 proximal promoter activity via two consensus Sp1-binding
sites (48, 49). How (or if) TSC-22 may act to enhance basal Sp1
transcriptional activity is unknown. Clearly, a major goal of future
work will be to identify genes that are directly regulated by TSC-22
and determine the mechanism(s) by which TSC-22 modulates the expression
of these genes. Determining TSC-22 target genes in the intestine will
lead to much better understanding of the exact role of this protein
during colon epithelial differentiation.
. It will be of interest to determine what
percentage of these colorectal tumors also contain mutations in
PPAR
. If this is not found to be a common occurrence, then our
present studies predict that treatment with activators of PPAR
may
provide a way to bypass the loss of normal TGF-
signaling that
occurs during the progression of these tumors.
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ACKNOWLEDGEMENTS |
---|
We thank Howard Crawford for help throughout this project, Chris Ferris for reviewing the manuscript, and Jian Tan and Sudhansu K. Dey for their generous help with the in situ hybridization studies.
![]() |
FOOTNOTES |
---|
* This work was supported in part by United States Public Health Service Grants RO1DK 47279 (to R. N. D.), P030 ES-00267-29 (to R. N. D.), and P01CA-77839 (to R. N. D.) and is supported by grants from the Shiseido Company of Japan, Ltd. (to L. A. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by grants from the American Cancer Society and the
National Institutes of Health.
Recipient of a Veterans Affairs Research Merit Grant, a
Mina C. Wallace Professor of Cancer Prevention, and supported by the T. J. Martell Foundation and National Colorectal Cancer
Research Alliance. To whom correspondence should be addressed:
Dept. of Medicine/GI, MCN C-2104, Vanderbilt University Medical Center, 1161 21st Ave. South, Nashville, TN 37232-2279. Tel.: 615-322-5200; Fax: 615-343-6229; E-mail: raymond.dubois@vanderbilt.edu.
Published, JBC Papers in Press, December 4, 2002, DOI 10.1074/jbc.M208076200
2 T. Soma, C. Dohrmann, G. Bi, J. Brissette, and L. Raftery, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
PPAR, peroxisome proliferator-activated receptor
;
TGF-
, transforming
growth factor-
;
TSC-22, transforming growth factor simulated
clone-22;
RD 1 and 2, repressor domains 1 and 2;
DRB, 5,6-dichloro-
-D-ribofuranosyl-benzimidazole;
wt, wild-type;
dn, dominant negative;
HA, hemagglutinin.
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REFERENCES |
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