 |
INTRODUCTION |
The TGF-
1 family of
proteins number >25 and regulate cell growth and differentiation as
well as morphogenesis and angiogenesis (1, 2). There are three
mammalian isoforms, TGF-
1, TGF-
2, and TGF-
3, which are
structurally very similar with nine conserved cysteines. The TGF-
s
belong to a superfamily of structurally related proteins including the
activins, inhibins, and bone morphogenic proteins (3). The TGF-
s
induce diverse biological responses by binding to the high affinity
receptors T
RI (53 kDa) and T
RII (75 kDa), which function as a
heterodimer. Both receptors have a cysteine-rich extracellular domain,
one transmembrane segment, and a cytoplasmic tail that includes a
serine/threonine kinase domain (4, 5). Constitutively phosphorylated
T
RII binds TGF-
1, which then recruits T
RI into the complex.
T
RI is transphosphorylated by T
RII and propagates the signal by
its kinase activity to downstream substrates (6). Two other
cell-surface TGF-
-binding proteins are the type III receptors
betaglycan and endoglin, which modulate cellular responses to TGF-
,
but have no signaling sequences. Betaglycan and endoglin may function
by regulating TGF-
access to T
RII (7-9). T
RIII receptors are
not found in every TGF-
-responsive cell and are down-regulated
during myoblast differentiation into myotubes (10).
The chief mediators of TGF-
signaling are the SMAD family of
structurally related proteins. Receptor-activated SMAD proteins form
heterotrimeric complexes and translocate to the nucleus, where they
interact with other transcription factors to drive TGF-
1-induced
transcription (11). Transcription factors interacting with SMAD
proteins include FAST-1 and FAST-2 (12, 13) and the c-Jun/c-Fos
heterodimer (14). The cooperation of SMAD proteins with other
transcription factors suggests that TGF-
1 induces multiple parallel
signaling pathways. Additional reported members of the TGF-
signaling pathways include the SMAD-interacting protein SARA and
related proteins (15), Ras (16-18), the Raf homolog TAK-1 (19),
TAK-1-associated proteins TAB-1 and TAB-2 (20), and the FK506- and
rapamycin-binding protein FKBP-12 and the
-subunit of
farnesyltransferase (21-23).
Several investigators have reported that expression of oncogenic
Ras confers resistance to the growth inhibitory properties of TGF-
(24-27). In mouse mammary epithelial cells, oncogenic Ras was
shown to constitutively activate the MAPKs ERK1 and ERK2. Activated
ERK1/ERK2 phosphorylate multiple sites within the linker regions of
SMAD2 and SMAD3, decreasing, but not totally eliminating, SMAD
translocation into the nucleus following TGF-
stimulation (28).
Oncogenic Ras, by this mechanism, eliminates the antimitogenic activity of TGF-
. However, treatment of the same oncogenic
Ras-transformed cells with TGF-
1 causes an
epithelial-to-fibroblastoid conversion to a more invasive phenotype
(29), showing that these cells maintained some TGF-
signaling
pathways, which mediated tumor aggressiveness. In the mouse skin model
of chemical carcinogenesis, one Ha-ras gene is activated by
mutation and the normal is allele lost, causing the cells to undergo a
morphological transformation to a highly invasive spindle phenotype
that can be induced in synergy with TGF-
1 (30).
We now report another system in which oncogenic Ras and TGF-
1
work together to enhance tumorigenicity and also report initial studies
on the mechanism of this response. Goblet cells compose ~20% of the
colon epithelial cell population and are resistant to growth inhibition
by TGF-
1 in vivo, becoming enriched relative to other
colon epithelial cells in mice injected with recombinant TGF-
1 (31).
Colon goblet cell lines are also resistant to the growth inhibitory
effects of TGF-
1 (32, 33). We now show that oncogenic Ras
up-regulates post-translational modification of the type III TGF-
receptor in the HD6-4 colon goblet cell line. Unexpectedly, TGF-
1
induces cell proliferation in the presence of oncogenic
Ki-ras and, in parallel, down-regulates the Cdk inhibitor p21Cip1/Waf1 and the tumor suppressor phosphatase PTEN.
 |
EXPERIMENTAL PROCEDURES |
Materials--
125I-TGF-
1 and
[3H[TdR were obtained from PerkinElmer Life Sciences.
Human recombinant TGF-
1 was from R&D Systems. Protein A-Sepharose was obtained from Amersham Pharmacia Biotech. Polyvinylidene difluoride transfer paper (Immobilon-P) was from Millipore Corp. Antibody TED1 to
PTEN was the kind gift of Dr. Hong Sun (Department of Genetics, Yale
University School of Medicine). Antibodies to T
RI/Alk5, TGF-
1,
and T
RII were purchased from Santa Cruz Biotechnology; antibodies to
phospho-SMAD2 and total SMAD2 were from Upstate Biotechnology, Inc.;
antibody to betaglycan/T
RIII was from R&D Systems; and mouse
monoclonal IgG2a clone 70 to p21Cip1 and
anti-phosphotyrosine monoclonal antibody PY69 were purchased from
Transduction Laboratories. Phosphorothiolated
oligodeoxynucleotides were a gift of Dr. Brett Monia (Isis
Pharmaceuticals, Carlsbad, CA). Isis-13177 is a 20-mer of random
sequence, and Isis-6957 is a 20-mer targeted to the 5'-untranslated
region of Ki-Ras (CAG-TGC-CTG-CGC-CGC-GCT-CG). This sequence is found
within the promoter region of the c-Ki-ras2 gene cloned into
pMiKVal12 (see below) ~60 bp upstream of the translational start
site. Endoglycosidase F (N-glycosidase F-free), also known
as endo-
-N-acetylglucosidase F, was purchased from Roche
Molecular Biochemicals. All other reagents, including chondroitinase ABC and heparitinase III, were from Sigma.
Antisense Oligonucleotide Treatment--
Treatment of
Ki-ras transfectants was essentially as described (34).
Cells were seeded at 2 × 105/well in six-well plates.
48 h later, the cells were washed with prewarmed serum-free
insulin transferrin/selenious acid-supplemented DMEM and
then incubated in this medium with a fixed ratio of oligonucleotide to
Lipofectin (2.4 µl of Lipofectin/100 µM
oligonucleotide) for 4 h. The oligonucleotide-containing medium
was then replaced with normal growth medium, and growth was continued
for 48 h to allow Ras turnover plus reduced Ras mRNA levels to
result in reduced Ras protein levels (34).
RT-PCR and Sequencing--
Total RNA was isolated from monolayer
cultures with TRIzol (Life Technologies, Inc.) according to the
supplier's manual. RNA was treated with DNase I (amplification-grade;
Life Technologies, Inc.) and RT-PCR was performed on a PerkinElmer Life
Sciences DNA Thermal Cycler 480 using a Promega Access RT-PCR kit as
follows and using the primers and conditions described previously (31). The amplification products were analyzed by ethidium bromide-agarose gel electrophoresis. The primer set used for RT-PCR of betaglycan mRNA yielded an expected amplification product of 592 bp, whereas primers for glyceraldehyde-3-phosphate dehydrogenase from
CLONTECH gave a product of 452 bp.
Cell Culture and Growth Studies--
Derivation of the oncogenic
Ki-ras and wild-type Ki-ras transfectants was as
described (35). All cell lines were maintained in low glucose DMEM
buffered with 25 mM Hepes (Atlanta Biologicals, Inc.) and
containing 7% fetal bovine serum as described (32). Cell density was
carefully controlled in these experiments. Within 1 day of growth to
confluency, cells were plated at one-third confluent density and then
used 2 days later. Serum-free transferrin/selenious acid-supplemented
DMEM was used for the TGF-
1 growth experiments (33).
TGF-
1-treated cells were less adherent, so both adherent and
floating cells were analyzed for [3H[TdR incorporation
after 3 h of labeling as described (33). Parallel wells that were
not seeded with cells were washed to remove nonspecifically bound
label, which was subtracted from all experimental values.
Transient Transfection of Betaglycan--
The c-Myc-tagged rat
betaglycan expression plasmid pCMV-mycBGL was received from J. Massague
(7) and subcloned into the retroviral vector pLXSN
(CLONTECH). The recombinant retrovirus was used to
infect mutant Ki-ras transfectant cells and control cells.
Immunoprecipitation of T
RIII following
[35S]Methionine Labeling--
Cells were incubated with
100 µCi/ml [35S]methionine in methionine-free DMEM for
6 h before lysates were prepared. 500 µg of cell lysates were
incubated overnight at 4 °C with 2 µg of anti-T
RIII antibody
and 40 µl of protein G Plus-agarose (Santa Cruz Biotechnology), pelleted at 2500 rpm, and washed three times with lysis buffer. After
the final wash, the pellet was mixed with 50 µl of SDS sample buffer
and boiled in a water bath for 5 min. Aliquots were analyzed by
SDS-PAGE, and T
RIII was detected by Western blotting using anti-T
RIII antibody and horseradish peroxidase-conjugated rabbit anti-goat IgG (Zymed Laboratories Inc.) and ECL.
Digestion with Endoglycosidase F--
Cell lysates were diluted
1:5 with detergent-free lysis buffer and then incubated for 36 h
with chondroitinase ABC (2 units/ml), heparitinase (2 units/ml), and
N-glycosidase F (5 units/ml) before SDS-PAGE and Western
blot analysis.
Immunodetection--
Cells grown in complete medium were lysed
in buffer containing 25 mM Tris (pH 7.4), 1% Triton X-100,
20 µg/ml leupeptin, 20 µg/ml aprotinin, 0.5 mM
phenylmethylsulfonyl fluoride, 200 µM sodium
orthovanadate, and 20 mM sodium fluoride. Depending on the
experiment, 30-70 µg of cell lysate proteins were blotted onto
polyvinylidene difluoride membranes after separation by SDS-PAGE. The
blots were blocked in blocking buffer (Tris-buffered saline containing
0.05% Tween 20 and 4% nonfat dry milk) for 1 h at room temperature. Primary antibodies were used in Tris-buffered saline containing 0.05% Tween 20 and 1% dry milk at the indicated
concentrations (anti-p21Cip1/Waf1, 1 µg/ml; anti-T
RI,
1 µg/ml; anti-T
RII, 1 µg/ml; and anti-T
RIII (betaglycan), 1 µg/ml) and dilutions (1:1000 TGF-
1, 1:1000 PTEN, 1:1000
phospho-SMAD2 and total SMAD2, and 1:1000 phosphotyrosine) and then
incubated for 2-3 h with primary antibody, and proteins were
subsequently detected by ECL (Amersham Pharmacia Biotech). For
p21Cip1 blots, cells were lysed in radioimmune
precipitation assay buffer, and blocking was performed in 1% dry milk
plus 4% bovine serum albumin. For SMAD2 blots, the first antibody was
diluted in 5% bovine serum albumin, and the second in 2.5% dry milk.
Affinity labeling of TGF-
receptors with
125I-TGF-
1 was performed essentially as described
(33).
Cell-surface Biotinylation and Immunoprecipitation of T
RI and
T
RII--
Cell-surface proteins were biotinylated essentially as
described by the vendor (Amersham Pharmacia Biotech). Briefly,
exponentially growing cells were washed once with cold
phosphate-buffered saline and once with 40 mM sodium
bicarbonate (pH 8.6). Cells were then incubated for 30 min at 4 °C
in 40 mM bicarbonate buffer containing 50 µl of biotin
reagent/ml of buffer, 4 ng/ml TGF-
1, and 1 mg/ml bovine serum
albumin (fatty acid-free). Following two washes with cold
phosphate-buffered saline, cells were lysed in 25 mM Tris buffer containing 1% Triton X-100, 20 µg/ml leupeptin, 20 µg/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride, 200 µM sodium orthovanadate, and 20 mM sodium
fluoride. 300 µg of each cell lysate were incubated overnight at
4 °C with 200 µl of streptavidin coupled to agarose beads (Sigma).
The beads were then washed extensively and resuspended in SDS-PAGE
sample buffer. Cell-surface T
RI and T
RII were immunoprecipitated
with anti-T
RII antibody C-16 with or without T
RII peptide C-16
(Santa Cruz Biotechnology) or with anti-T
RI antibody R-20 (Santa
Cruz Biotechnology)Biotechnology; antibodies were bound with
horseradish peroxidase-streptavidin after washing and then detected
with ECL.
Statistical Analysis--
Calculations measuring the difference
between two means were performed by the t test.
 |
RESULTS |
Oncogenic Ki-ras Transfectants Gain Response to TGF-
1--
The
Ki-rasG12V transfectant cloned sublines V, V1,
and V2 were isolated following stable transfection of HD6-4 colon
carcinoma cells with a mini-gene construct of the cellular
Ki-ras4B gene mutated at codon 12 to valine. Three stable
sublines (G, G2, and G3) were isolated following transfection with the
wild-type mini-gene construct of cellular Ki-ras4B (35, 36).
The Ki-rasG12V transfectant lines expressed
oncogenic Ki-Ras protein, whereas the wild-type
Ki-ras transfectant G lines expressed more wild-type Ki-Ras
protein as shown by Western blotting with Ki-Ras-specific and
pan-RasVal12-specific antibodies (35).
The parental line is not responsive to TGF-
1 (31-33), but
unexpectedly became TGF-
1-responsive following expression of
oncogenic Ki-Ras protein. Ki-rasG12V
transfectant V cells exhibited a rapid increase in tyrosine
phosphorylation of an unknown cellular protein of ~60 kDa after
addition of TGF-
1, whereas no increase in tyrosine phosphorylation
of this or any other protein was found in control cells treated with
TGF-
1 (Fig. 1A). The 60-kDa
protein showed increased tyrosine phosphorylation following 5 and 15 min of TGF-
1 treatment (Fig. 1A, arrow),
whereas the abundance of phosphotyrosine in a 200-kDa protein remained constant, demonstrating equal loading. The chief mediators of TGF-
signaling are the SMAD family of structurally related proteins. Upon
TGF-
receptor activation, T
RI activates phosphorylation of SMAD2
and/or SMAD3, each of which can form an association with the common
mediator SMAD4 and then translocate to the nucleus and mediate
transcription (reviewed in Refs. 11 and 37). SMAD2 is activated by
phosphorylation of serines 465 and 467 (38). 30 min of TGF-
1
treatment caused a striking increase in the activation of
phosphorylation of SMAD2 in the oncogenic Ki-ras V
transfectant, but no activation in control cells (Fig. 1B).
A slight increase in the total amount of SMAD2 and SMAD3 in the
Ki-ras V transfectant was also detected by Western blotting.
Thus, expression of mutant Ki-Ras protein unexpectedly restored an
initial step in a TGF-
signaling pathway in these cells.

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 1.
Ki-rasG12V
transfectant cells respond to TGF- 1, but
wild-type ras cells do not. A,
parallel cultures of Ki-rasG12V transfectant V
cells and parental wild-type ras cells
(RasWT) were treated with 8 ng/ml TGF- 1 for 0, 5, 15, or 30 min, and cell lysates were prepared and analyzed by Western
blotting with anti-phosphotyrosine (P-Tyr) antibody. The
arrow marks a protein exhibiting an increase in
phosphotyrosine content only in the Ki-rasG12V
transfectant V cells. A 200-kDa protein exhibited similar
phosphotyrosine content at all time points in both cell types.
B, parallel cultures of Ki-rasG12V
transfectant V cells and wild-type Ki-ras transfectant G
cells were treated with 1 ng/ml TGF- 1 for 30 min before cell lysis
and Western blotting with antibody directed to SMAD2 phosphorylated at
serines 465 and 467 (P-Smad2) and with antibody to total
SMAD2/SMAD3. The lower panel shows cellular proteins of
55-60 kDa after the Western blot was stained with Coomassie Blue to
demonstrate equal loading and transfer. IB,
immunoblotting.
|
|
Response to TGF-
1 by Oncogenic Ki-ras Transfectants Consistent
with Tumor Progression--
The Ki-rasG12V
transfectant lines were more tumorigenic in vivo and more
invasive in vitro than the control cells (35, 36). Mutant
Ki-Ras caused aberrant post-translational glycosylation of
1 integrin, loss of cellular adhesion to the
extracellular matrix, blocked cell polarization, and decreased
cell-cell adhesion properties through up-regulation of carcinoembryonic
antigen and down-regulation of N-cadherin (35, 36). These
decreases in attachment to the extracellular matrix and intercellular
adhesion provide one basis for selection for Ki-ras
mutations in colon cancers.
We had noted in past experiments that certain highly aggressive or
metastatic colon cancer cells responded to TGF-
1 by increased cell
growth (39-42) and wondered if the increased aggressiveness of the
Ki-rasG12V transfectant cells in vivo
reflected increased growth in response to autocrine or exogenous
TGF-
1. TGF-
1 induced a statistically significant,
dose-dependent increase in growth up to 2-fold in Ki-rasG12V transfectant V cells, whereas
TGF-
1 induced no change in proliferation of parental cells grown in
parallel (Fig. 2A, upper
panel). The p value for mean growth ± 1 ng/ml
TGF-
1 was <0.01, whereas the p values for growth ± 2 and 4 ng/ml TGF-
1 were both <0.001, all statistically significant
differences. This growth response to TGF-
1 was generalized by
assaying two additional Ki-rasG12V transfectant
cell lines, V1 and V2, and three other control lines at the optimal
TGF-
1 concentration (Fig. 2B, upper panel).
TGF-
1 at 4 ng/ml increased [3H[TdR incorporation 1.5- to >2-fold in V1 and V2 cells, with statistically significant
p values of <0.0005. In contrast, TGF-
1 had no effect on
cycling of wild-type Ki-ras transfectant G2 and G3 cells or the parental line with endogenous wild-type Ki-ras (Fig.
2B).

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 2.
TGF- 1 stimulates
proliferation of Ki-rasG12V transfectant
cells and down-regulates levels of the Cdk inhibitor p21 and the tumor
suppressor PTEN. A: upper panel, HD6-4 colon
carcinoma cells stably transfected with
Ki-rasG12V transfectant V cells and parental
cells expressing only wild-type ras
(RasWT) were treated for 54 h with increasing
concentrations of TGF- 1 in serum-free transferrin/selenious
acid-supplemented DMEM and then incubated with [3H[TdR
for 3 h before analysis to measure cell proliferation. Both
attached cells and loosely adherent cells were collected for this
assay. All data are the mean of two experiments, each performed in
triplicate. The t test demonstrated that the proliferation
of TGF- 1-treated V cells was statistically greater than that of
TGF- 1-treated control cells, with p values of <0.01,
0.001, and 0.001 for TGF- 1 levels of 1, 2, or 4 ng/ml, respectively.
Error bars are shown only if the S.E. is >5%.
Lower panel, TGF- 1 decreases the abundance of
p21 and PTEN in Ki-rasG12V transfectant V cells.
rasG12V and wild-type ras cells were
treated for 54 h with increasing concentrations of TGF- 1 of 0, 1, 2, 4, and 8 ng/ml, and then the abundance of p21Cip/Waf1
and PTEN in cell lysates was determined by Western blotting. The
lower panel shows cellular proteins of ~44 kDa detected in
this immunoblot by staining with Coomassie Blue to demonstrate equal
loading. B: upper panel, TGF- 1 stimulates
proliferation of two other Ki-rasG12V
transfectants. Four other stable ras transfectants, two with
mutant Ki-rasG12V (V1 and V2 cells) and two with
wild-type Ki-ras (G2 and G3 cells), and the parental line
were treated with 4 ng/ml TGF- 1 or left untreated, and
[3H[TdR incorporation for each cell line was determined
as described for A. All data are the mean of two
experiments, each performed in triplicate. The t test
demonstrated that the proliferation of TGF- 1-treated V1 and V2 cells
was statistically greater than that of untreated V1 and V2 cells, with
p values <0.0005 for each line. Lower
panel, Western blots for the Cdk inhibitor p21 and the tumor
suppressor PTEN and a double protein band of ~44 kDa detected by
Coomassie Blue staining of the Western blot to demonstrate equal
loading.
|
|
The mechanism of this unexpected proliferative response to TGF-
1 was
explored by assaying two proteins associated with tumorigenicity: the
Cdk inhibitor p21Cip1/Waf1 (43, 44) and the tumor
suppressor phosphatase PTEN (45, 46). p21Cip1/Waf1 inhibits
cell cycling by targeting most Cdk-cyclin complexes, binding to them
and inhibiting their kinase activity. PTEN is a phospholipid
phosphatase, deleted or mutated in a wide variety of tumors (46). Loss
of PTEN activity would up-regulate the activity of the serine/threonine
kinase Akt, which is known to play an important role in generating cell
survival signals (47-49). Ki-rasG12V
transfectant V cells and control cells were treated for 2 days with 0, 1, 2, 4, or 8 ng/ml TGF-
1, and then cell lysates were blotted for
abundance of p21 and PTEN (Fig. 2A, lower panel). In control cells with wild-type Ki-ras, TGF-
1 caused no
alteration in the levels of either protein. However, in duplicate
experiments, TGF-
1 induced a dose-dependent decrease in
the abundance of the Cdk inhibitor p21Cip1/Waf1, with the
optimal growth-stimulating concentration of 4 ng/ml TGF-
1 reducing
p21 levels to 16% of the control. PTEN levels were decreased 6-fold by
2, 4, or 8 ng/ml TGF-
1 only in the mutant Ki-Ras transfectant V
cells. Coomassie Blue staining of the blot demonstrated equal loading
(Fig. 2A, lower panel).
The generality of these responses was determined by assaying several
more cell lines. Levels of p21Cip1/Waf1 and PTEN were
examined in two other oncogenic Ki-ras transfectant lines
(V1 and V2), two wild-type Ki-ras transfectant lines (G2 and
G3), and the parental line with endogenous wild-type ras. Treatment with the optimal concentration of 4 ng/ml TGF-
1 decreased p21 levels in V1 and V2 cells expressing mutant Ki-Ras by 52 and 90%,
respectively, whereas no decreases in p21 levels were observed in G1
and G3 lines with wild-type ras (Fig. 2B,
lower panel). Treatment with 4 ng/ml TGF-
1 decreased PTEN
levels in V1 and V2 cells expressing mutant Ki-Ras by 50 and
30%, respectively, in duplicate experiments. In contrast, no decreases
were observed in PTEN levels in G2 and G3 cells expressing only
wild-type Ki-ras. Thus, TGF-
1 acts like a mitogen in
mutant Ki-ras-transformed colon carcinoma cells,
possibly by decreasing the abundance of the Cdk inhibitor
p21Cip1/Waf1. The loss of PTEN expression with TGF-
1
treatment may contribute to the aggressive growth of each
Ki-rasG12V transfectant cell line in
vivo, as cells can respond to TGF-
1 from autocrine or paracrine
sources (35). These experiments demonstrate that oncogenic
Ki-ras can confer a more aggressive, tumorigenic phenotype
in colon cancer cells by altering their response to TGF-
1.
The Cdk inhibitor p21 is induced by TGF-
1 acting at the
transcriptional level in several cell types, including (most if not all) epithelial cells (50, 51), but decreases in p21 abundance are seen
when cells are treated with a mitogen. TGF-
1 has also been reported
to rapidly down-regulate expression of PTEN at the transcriptional
level in human keratinocyte HaCaT cells (45). Ongoing studies are
exploring the mechanisms for these changes. In the following studies,
we provide the mechanism for the gain of response to TGF-
1 by
oncogenic ras.
Functional TGF-
Receptors Induced in Ki-rasG12V
Transfectants without Changes in T
RI, T
RII, or TGF-
1 Abundance
or Mobility--
Functional T
RI, T
RII, and T
RIII receptors
had not been detected in parental cells expressing wild-type
Ki-ras in earlier studies using cross-linking of
125I-TGF-
1 (33, 31). TGF-
1-binding proteins on the
surface of viable cells were chemically cross-linked with
125I-TGF-
1 and then molecularly sized by SDS-PAGE and
detected by autoradiography. Functional T
RI and T
RII and a
polydisperse band of higher molecular mass TGF-
1-binding proteins of
the size of T
RIII were detected in each
Ki-rasG12V transfectant line (V, V1, and V2). In
contrast, the parental line HD6-4 expressing only endogenous wild-type
Ki-ras (Fig. 3, left
panel, WT lane) and the wild-type Ki-ras
transfectant G cell line (right panel) exhibited no
detectable TGF-
1 binding. Identical amounts of labeled TGF-
1 were
detected on each lane in the autoradiograph, demonstrating equal
loading. The parental cells displayed no binding of
125I-TGF-
1, although neither the T
RII nor T
RI
receptor was mutated, and these cells exhibited cell-surface T
RI and
T
RII proteins at levels equal to those seen in TGF-
1-responsive
cells, indicating that receptor localization was not altered (31).
Thus, functional T
RI, T
RII, and T
RIII were detected in each of
three Ki-rasG12V transfectant lines, but in
neither control line.

View larger version (77K):
[in this window]
[in a new window]
|
Fig. 3.
Functional TGF-
receptors found after stable transfection of
Ki-rasG12V in each of three lines.
Viable parental HD6-4 cells (wild-type (WT)),
rasG12V cells (V, V1, and V2), and stable
wild-type Ki-ras transfectant G cells were cross-linked with
125I-TGF- 1, and then cell lysates were analyzed by
autoradiography following size fractionation by 5-8% gradient
SDS-PAGE and electroblotting to polyvinylidene difluoride membrane. The
positions of T RI, T RII, and the region where polydisperse
T RIII migrates are indicated. Non-cross-linked
125I-TGF- 1 is seen at the bottom of the blot.
|
|
Possibly oncogenic Ki-Ras could increase the ability of TGF-
receptors to bind 125I-TGF-
1 by altering the level of
autocrine TGF-
1. However, no difference in the level of mature
TGF-
1 was discerned between the Ki-rasG12V
transfectant V cells and parental cells (Fig.
4). Thus, oncogenic Ki-Ras protein did
not alter the abundance or electrophoretic mobility of autocrine
TGF-
1. No differences in cellular abundance, cell surface levels, or
electrophoretic mobility of T
RI or T
RII were detected in the two
cell types, expressing either wild-type or mutant Ki-Ras (Fig.
5). Total T
RI levels were assayed by
Western blotting (Fig. 5A), and cell-surface T
RI levels
were assayed by immunoprecipitation following biotinylation (Fig.
5C). Total T
RII levels were assayed by
immunoprecipitation (Fig. 5B), and cell-surface T
RII
levels were assayed by immunoprecipitation following biotinylation
(Fig. 5C). Each experiment was controlled by absorption of
the antibody with specific peptides corresponding to either T
RI or
T
RII. The biotinylation experiment demonstrated that equal amounts
of the T
RI and T
RII proteins reached the cell surface of cells
expressing either wild-type or mutant Ki-Ras. Thus, the increased
binding of 125I-TGF-
1 in the oncogenic Ki-ras
transfectant cells (Fig. 3) was not a result of increased transport of
receptor proteins to the cell surface. These results, taken together,
demonstrate that expression of oncogenic Ki-Ras protein did not alter
the abundance, cell-surface expression, or electrophoretic mobility of
either T
RI or T
RII, although these receptors regained the ability
to bind exogenous 125I-TGF-
1 when oncogenic Ki-Ras
protein was expressed.

View larger version (56K):
[in this window]
[in a new window]
|
Fig. 4.
Stable transfection of oncogenic
Ki-ras does not alter abundance or migration of
TGF- 1. Left panel, Western
blot for TGF- 1 in parental wild-type ras cells
(RasWT) and rasG12V
transfectant V cells; right panel, Coomassie Blue-stained
Western blot to demonstrate equal loading. IB,
immunoblotting.
|
|

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 5.
Stable transfection of oncogenic
Ki-ras does not alter total abundance, cell-surface
abundance, or electrophoretic mobility of T RI
or T RII. A, shown are parallel
Western blots for T RI in lysates from parental HD6-4 cells
(RasWT) and rasG12V
transfectant V cells. The + lanes were blotted in the
presence of the immunizing receptor I peptide to demonstrate
specificity of the blotting. B, T RII was detected by
immunoprecipitation (IP) of lysates from parental wild-type
ras cells and rasG12V V transfectant
cells. A duplicate wild-type sample was immunoprecipitated in the
presence of the receptor II peptide immunogen as a control, The
arrowhead indicates T RII. C, proteins at the
cell surface of parental wild-type ras cells and
rasG12V transfectant V cells were biotinylated,
and then T RI and T RII were immunoprecipitated and then detected
by ECL. In the + lane, immunoprecipitation was performed in
the presence of the immunizing receptor I or II peptide to demonstrate
specificity of the immunoprecipitation. Arrowheads indicate
T RI (RI) and T RII (RII). Similar levels of
nonspecific bands in each blot demonstrate equal loading.
IB, immunoblotting.
|
|
Restoration of T
RIII function by itself might explain the ability of
T
RI and T
RII to bind TGF-
1 since the parental cells exhibit
incomplete post-translational modification of T
RIII (31). T
RIII
functions by concentrating TGF-
in the cell periphery and presenting
it to T
RII (8, 9, 52). T
RII then forms a complex with T
RI and
phosphorylates T
RI, transmitting the TGF-
1 signal (53).
Therefore, expression of mutant Ki-Ras was closely correlated with gain
of TGF-
receptor function.
Increased Post-translational Modification of Endogenous
Betaglycan/T
RIII in Oncogenic Ki-rasG12V
Transfectants--
The major form of T
RIII found in HD6-4 colon
carcinoma cells is betaglycan, whose core protein migrates at ~110
kDa (31). Very little post-translational modification of betaglycan
occurs in parental cells with wild-type ras (31). In
TGF-
1-responsive colon carcinoma cells, in contrast, betaglycan
migrates as more modified forms at 120 and 140 kDa as well as a
heterogeneous diffuse group of species above 200 kDa (31).
Post-translational glycosylation of the integrin
1-chain
was found to be aberrant in each of the oncogenic Ki-ras
transfectants (35), suggesting that post-translational glycosylation of
betaglycan might also be altered. Oncogenic forms of ras
genes have been implicated in modulating various Golgi acetylglucosaminyltransferases in different cell types (54, 55).
Western blotting for T
RIII revealed new high molecular mass bands
for each of the three Ki-rasG12V transfectants,
but not for the wild-type Ki-ras G cells (data not
shown), indicating greater post-translational modification of
betaglycan. Thus, oncogenic Ki-Ras might up-regulate an
acetylglucosaminyltransferase or a galactosyltransferase, which would
modify betaglycan and restore its biological function.
The post-translational modifications of endogenous betaglycan were
analyzed in Ki-rasG12V transfectant V cells by
immunoprecipitation after prelabeling cellular proteins with
[35S]methionine. Betaglycan forms were size-fractionated
on gradient SDS-PAGE and detected by autoradiography. In control cells
with wild-type ras, betaglycan/T
RIII migrated as a core
peptide of 110 kDa (Fig. 6A,
left panel) and one higher molecular mass, but low abundant
diffuse band (arrows). Thus, little post-translational modification occurred in control cells, as reported previously (31). In
contrast, three higher molecular mass betaglycan species in addition to
the core protein were detected in lysates from Ki-rasG12V transfectant cells (Fig.
6A, left panel, arrowheads).
Therefore, at least some of the high molecular mass
125I-TGF-
1-binding proteins detected in each
Ki-rasG12V transfectant line (Fig. 3) were
post-translationally modified betaglycan/T
RIII.

View larger version (60K):
[in this window]
[in a new window]
|
Fig. 6.
More abundant post-translational modification
of endogenous T RIII in
rasG12V cells. A:
left panel, wild-type ras
(RasWT) and rasG12V (P and V
cells) were prelabeled with [35S]methionine, and then
T RIII was immunoprecipitated, size-fractionated by SDS-PAGE, and
autoradiographed. The upper region of the autoradiograph is shown,
including the core peptide (arrow). There are three
polydisperse bands of post-translationally modified T RIII in
rasG12V transfectant cells
(arrowheads on the right), but only one major band in the
control wild-type ras cells (arrowhead on the
left). Right panel, the lysates were treated with
N-glycosidase F, heparitinase, and chondroitinase ABC to
remove proteoglycan chains and glycosylations before
immunoprecipitation of T RIII. Post-translational modifications of
T RIII were lost, leaving only similar levels of core protein.
B, semiquantitative RT-PCR demonstrated that there are
similar levels of betaglycan mRNA in rasG12V
transfectant V, V1, and V2 cells compared with wild-type ras
transfectant G, G2, and G3 cells. In the three control lanes, no
mRNA was added (first lane), or no reverse transcriptase
was added to the mRNA preparations (second and
third lanes). The positions of the betaglycan 592-bp PCR
product and the 452-bp internal control glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) product are indicated.
Degly, deglycosylated.
|
|
We next questioned whether the post-translational modification of
betaglycan induced by mutant Ki-Ras was similar to that seen in other
TGF-
1-responsive cells. In such cells, the betaglycan core protein
is heavily modified by glycosaminoglycan groups and by
N-glycosylation (56). The betaglycan in V cells and in
parental cells was chemically deglycosylated with
N-glycosidase F, and proteoglycan groups were removed by
digestion with heparitinase and chondroitinase ABC (Fig. 6A,
right panel). These treatments have been shown to reduce
betaglycan to its protein core (56). After treatment, equal amounts of
betaglycan core protein were immunoprecipitated from prelabeled V cells
and control cells, and the core proteins displayed equivalent
electrophoretic migration rates (Fig. 6A, left
and right panels). Thus, mutant Ki-Ras functioned at the
level of post-translational modification of betaglycan, just as mutant
Ki-Ras functioned in the same cells to modulate post-translational
modification of
1 integrin (35).
These data indicated that betaglycan mRNA levels should be similar
in mutant Ki-Ras and control cells. Similar amounts of betaglycan
mRNA were detected in Ki-rasVal12
transfectant and control cells by semiquantitative RT-PCR (Fig. 6B), consistent with the equal levels of betaglycan core
peptide exhibited in each cell line. These data suggest that mutant
Ki-Ras functions to increase betaglycan modification, not to increase the abundance of the betaglycan core protein.
More Abundant Post-translational Modification of Transiently
Transfected Betaglycan in Ki-rasG12V Transfectant
Cells--
To confirm that betaglycan was highly modified following
translation in Ki-rasG12V transfectant cells,
betaglycan tagged with a c-Myc epitope was inserted into a retroviral
vector. Both Ki-rasG12V transfectant V cells and
control cells expressing wild-type Ki-ras were infected with
this recombinant retrovirus. The predominant betaglycan species
immunoprecipitated from control cells was the core peptide (Fig.
7). Therefore, very little
post-translational modification of betaglycan occurred in cells with
wild-type ras, consistent with the data on endogenous
betaglycan shown in Fig. 6A. Moreover, highly modified,
polydisperse betaglycan was synthesized in the
Ki-rasG12V transfectant V cells (Fig. 7), again
confirming the increased post-translational modification of endogenous
betaglycan (Fig. 6A). Control experiments were carried out
with the retroviral vector alone (data not shown) or without
the c-Myc antibody in immunoprecipitations (
lanes). Note that equal amounts of IgG were found in each
antibody lane, confirming the additional of equal amounts of antibody.
Therefore, betaglycan/T
RIII, whether transfected or endogenous, was
highly modified following translation in the
Ki-rasG12V transfectant cells, but not in
control cells expressing only wild-type Ki-ras.

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 7.
Exogenous betaglycan is highly modified in
rasG12V transfectants.
rasG12V (V clone) and parental wild-type
ras (RasWT) cell lines were infected with
either a recombinant retrovirus encoding a Myc-tagged betaglycan gene
(LXSN-Myc-BG) or a recombinant retrovirus encoding only the c-Myc
expression tag (LXSN-Myc). After 48 h of culture to allow
expression and post-translational modification of betaglycan, the
expressed betaglycan was immunoprecipitated (IP) by its
c-Myc tag and analyzed by Western blotting with anti-T RIII antibody.
The core betaglycan peptide was synthesized in both cell types
(myc-BG core), but was highly modified only in
rasG12V cells (myc-BG). Similar
levels of IgG were detected in each immunoprecipitate as controls.
Ab, antibody.
|
|
Antisense Oligonucleotides to Ki-Ras Decrease
Post-translational Modification of Betaglycan and Block Down-regulation
of p21 by TGF-
1--
The possibility was investigated that
betaglycan modifications occurred as a result of adaptation of
Ki-rasG12V transfectants to culture and were not
directly related to expression of mutant Ki-Ras protein. Therefore, the
expression of mutant Ki-rasG12V in V2 cells was
decreased by treatment with phosphorothiolated antisense
oligonucleotides targeted to Ki-Ras. This treatment almost completely
eliminated the expression of the mutant Ki-Ras protein in V2 cells
compared with cells treated in parallel with random sequence
oligonucleotides, as shown by Western blotting with a
pan-RasVal12-specific antibody (Fig.
8A, left panel).
Phosphorothiolated antisense oligonucleotides directed to mutant Ki-Ras
block the abnormal glycosylation of the integrin
1-chain
in V2 cells (35), so we expected that blocking mutant Ki-Ras would
alter betaglycan modification. The sharp decrease in mutant Ki-Ras
protein caused a marked decrease in the abundance of the most highly
modified betaglycan species in the Ki-ras antisense
oligonucleotide-treated cells (Fig. 8A, right
panel) compared with cells treated with random sequence
oligonucleotides. The decrease in betaglycan modification was dependent
on the concentration of phosphorothiolated antisense oligonucleotides,
with the greatest effect seen at 200 nM. Each of the
lysates exhibited similar amounts of core protein, demonstrating equal
loading. Therefore, the increased post-translational modification of
betaglycan/T
RIII found in each of the oncogenic ras
transfectant lines was due to expression of the mutant
Ki-ras gene.

View larger version (58K):
[in this window]
[in a new window]
|
Fig. 8.
Antisense oligonucleotides directed to Ki-Ras
greatly decreases expression of mutant Ki-RasVal12 protein,
decrease T RIII post-translational modification
in Ki-rasG12V cells, and block induction
of p21 by TGF- 1. A: left
panel, rasG12V transfectant V2 cells were
treated for 4 h with phosphorothiolated antisense oligonucleotides
or phosphorothiolated random sequence oligonucleotides or left
untreated (control lane) and then washed and cultured in
oligonucleotide-free medium for 48 h to allow Ras turnover plus
reduced Ras mRNA levels to result in reduced Ki-Ras protein.
Western blotting was performed with pan-RasVal12 monoclonal
antibody, which detects only the forms of the oncogenic Ras protein
mutated at codon 12 to valine. Right panel, T RIII was
immunoprecipitated (IP) after treatment with antisense or
random sequence oligonucleotides. During the last overnight incubation
in A, cells were prelabeled with
[35S]methionine, and then T RIII was
immunoprecipitated, size-fractionated by SDS-PAGE, and
autoradiographed. The upper region of the autoradiograph is shown, and
post-translationally modified T RIII (RIII) is indicated
as well as the faster migrating core peptide that was present at
similar levels in each immunoprecipitate. B,
rasG12V transfectant V2 cells were treated as
described for A with 200 nM antisense
oligonucleotides directed to Ki-Ras or 200 nM random
sequence oligonucleotides. After culture for 6 h in
oligonucleotide-free complete DMEM, cells were treated with 4 ng/ml
TGF- 1 in serum-free transferrin/selenious acid-supplemented DMEM for
44 h or left untreated. The abundance of the Cdk inhibitor p21 was
then analyzed by Western blotting. The abundance of a nonspecific
protein on the Western blot is shown (Protein level) to
demonstrate equal loading and transfer. IB, immunoblotting;
RasWT, wild-type ras cells.
|
|
We next tested whether blocking expression of the mutant
Ki-ras gene would also block response to TGF-
1. V2 cells
were treated with equal concentrations (200 nM) of either
phosphorothiolated antisense oligonucleotides to Ki-Ras or
phosphorothiolated random sequence oligonucleotides as described above
and then treated for 2 days with 4 ng/ml TGF-
1 or left untreated.
TGF-
1 down-regulated the abundance of the Cdk inhibitor
p21Cip1/Waf1 in V2 cells pretreated with random sequence
oligonucleotides in duplicate experiments analyzed by Western blotting
(Fig. 8B). However, the TGF-
1 response was blocked in V2
cells treated with antisense oligonucleotides to down-regulate mutant
Ki-Ras, and the abundance of p21Cip1/Waf1 was unchanged
(Fig. 8B). The abundance of a cellular protein slightly
larger than p21Cip1/Waf1 is also shown to demonstrate equal
loading and blotting. These experiments demonstrate that the increased
post-translational modification of betaglycan/T
RIII observed in each
Ki-rasG12V transfectant line and the response of
the Ki-rasG12V transfectant cells to TGF-
1
were caused by expression of the mutant Ki-ras gene.
 |
DISCUSSION |
T
RIII or betaglycan is the most abundant TGF-
-binding
protein in a number of cell types. Betaglycan has a small cytoplasmic domain with no consensus signaling motif (7, 9), so it is not believed
to directly transduce TGF-
signaling. However, in the absence of
T
RIII, only a small fraction of T
RII in myoblasts binds TGF-
1
with high affinity, whereas expression of T
RIII in these cells
converts the majority of T
RII molecules to high affinity TGF-
1
receptors (52). Thus, the major role of T
RIII is believed to be its
ability to concentrate TGF-
1 and present it to the signaling
receptors, T
RII and T
RI. The role of T
RIII is not limited to
binding exogenous TGF-
1. Stable transfection of T
RIII in MCF-7
breast cancer cells restores autocrine TGF-
1 signaling as well
(57).
In this study, we have shown that both functional, post-translationally
modified T
RIII and the biological response to TGF-
1 were restored
by stable expression of oncogenic Ki-rasVal12 in
HD6-4 colon carcinoma cells. Both responses were blocked by phosphorylated antisense oligonucleotides, which markedly reduced the
abundance of mutant Ki-Ras protein, proving that the changes in TGF-
receptors and response were caused by the mutant Ki-Ras protein. The
oncogenic Ki-rasVal12 cell lines had been shown
in earlier studies to have altered post-translational glycosylation of
1 integrin (35), so the post-translational modification
of T
RIII shown in the this study may also be glycosylation. TGF-
1
mediates glycosylation of
1 integrin in colon carcinoma
cells by activating Ras proteins (58). The TGF-
1/Ras pathway
functions to convert a partially glycosylated
1 integrin
precursor into the mature, fully glycosylated form and is blocked by
dominant-negative N17Ras (58). In like fashion, constitutive activation
of Ki-Ras in stable oncogenic Ki-ras transfectants may
up-regulate glycosyltransferases that modify T
RIII.
1-6-N-Acetylglucosaminyltransferase V is increased
severalfold in activity in rodent fibroblast lines transfected with the
oncogenic T24 Ha-ras gene (54). In addition, NIH3T3 cells
expressing the N-ras proto-oncogene exhibit 5-7-fold increases in the activities of
1-galactosyltransferase and
3-N-acetylglucosaminyltransferase, both of which
synthesize polylactosaminoglycan chains (55). What is the biological
significance of these observations? ras genes are mutated in
about half of all colon cancers. The colon goblet cell lineage
comprises 20% of the cells in the colonic crypt, so some of the
ras mutations that occur in the development of colon cancer
must take place in this lineage.
The parental HD6-4 cell line used in this study was shown
earlier to respond to very high levels of TGF-
1 (100 ng/ml),
20-100 times more than used in this study, by induction of
p21Cip1/Waf1 (31). Lower concentrations of 1-5 ng/ml
TGF-
1, which initiated responses in other colon carcinoma cells and
in the mutant Ki-Ras transfectants in this study, were ineffective on
the parental HD6-4 cells (31). Very high TGF-
1 levels of 100 ng/ml
could bypass the need for a functional T
RIII to bind and concentrate TGF-
1 and to present it to the signaling receptors, leading us to
hypothesize that, under these conditions, TGF-
1 bound directly to
T
RII. Supporting this hypothesis, expression of inducible dominant-negative T
RII blocked signaling caused by very high TGF-
1 levels. Thus, signaling did occur through the T
RII-mediated pathway. HD6-4 cells also responded to autocrine TGF-
1 by
1 integrin maturation, and this TGF-
response to
autocrine TGF-
1 was also blocked by inducible
dominant-negative T
RII (31). Therefore, before transfection with
mutant Ki-ras, HD6-4 cells had intact TGF-
signaling pathways. These intact pathways mediated induction of
p21Cip1/Waf1 in response to TGF-
1 and maturation of
1 integrin in response to autocrine TGF-
1.
These results demonstrate that cells expressing mutant Ki-Ras became
much more sensitive to added TGF-
1, and their response to TGF-
1
was altered. TGF-
1 was no longer capable of up-regulating levels of
p21Cip1/Waf1 in mutant Ki-Ras cells as it did in the
parental HD6-4 cell line (31) and, in fact, displayed the opposite
response to TGF-
1, decreasing levels of p21Cip1/Waf1. It
is not yet known whether the TGF-
1 effects on
p21Cip1/Waf1 are mediated at the transcriptional or
post-translational level. TGF-
1 is mitogenic in osteoblasts; and in
these cells, TGF-
1 stimulated degradation of the Cdk inhibitors p57,
p27, and p21 by the proteasome (59). Thus, TGF-
1 may induce rapid
turnover of p21Cip1/Waf1 in oncogenic Ki-ras
transfectants and, in so doing, increase their growth rate. In fact,
Ki-ras transfectants grow much more quickly than control
cells in athymic mice (35).
What is the basis for this switch in response to TGF-
1? The general
picture has emerged that TGF-
1 has a dual role in carcinoma development: TGF-
1 inhibits the proliferation of normal cells (60,
61) and benign tumor cells (62), but enhances malignancy at later
stages of tumorigenesis in cells that retain some response to TGF-
(40, 63-66). Supporting this model are studies showing that TGF-
1
inhibits benign skin tumor formation, but enhances progression of skin
tumors to invasive spindle carcinomas in carcinogen-treated transgenic
mice with keratinocyte-targeted expression of TGF-
1 (67). The
tumor-promoting effects of TGF-
1 are more evident when tumor cells
are highly aggressive. The metastatic potential of mammary
adenocarcinoma cells is increased by in vitro treatment with
TGF-
1 before injection into syngeneic rats (68). Highly aggressive
and metastatic U9 colon cancer cells use autocrine TGF-
1 to mediate
their growth and invasion (40). Decreasing TGF-
1 protein levels in
the metastatic U9 colon cancer cell line by antisense methodology
decreases both U9 cell metastasis to the liver and subcutaneous tumor
formation in a nude mouse system, and the tumors that do arise regain
TGF-
1 expression (69). Also, levels of TGF-
1 protein within colon
cancers and in serum are enhanced in patients whose disease progresses
(70-73). Many other types of carcinomas, including pancreatic cancer,
gastric cancer, endometrial cancer, breast cancer, gliomas, and
osteosarcomas, also respond to TGF-
1 with disease progression
(2).
The mechanism of this switch in response to TGF-
1 is unknown, but
may include alterations in Ras. ras genes are mutated in ~30% of all cancers and about half of all colon cancers. In some cases, residual TGF-
signaling pathways may remain functional after
some TGF-
signaling pathways are blocked by mutations in T
RII or
SMAD proteins (74-76). Such residual pathways may mediate epithelial-to-mesenchymal transdifferentiation (77). In
ras-transformed cells, the TGF-
1 signaling cascade, which
leads to growth inhibition, is blocked by MAPK phosphorylation of
signal-transducing SMAD proteins (28). However, the
ras-transformed cells retain TGF-
response, demonstrating
that these signals are relayed either by residual SMAD activity or by a
SMAD-independent mechanism. In ras-transformed IEC
cells, TGF-
1 growth resistance is lost, but the cells retain some
TGF-
signaling systems, as the cells remain responsive to TGF-
1
stimulation of fibronectin synthesis (78). Likewise, in
ras-transformed mammary epithelial cells, TGF-
1 growth
inhibition is lost, but is replaced by the capacity to undergo
epithelial-to-mesenchymal transdifferentiation with increased cell
invasiveness (29).
Autocrine TGF-
1 (Fig. 4) may mediate an epithelial-to-mesenchymal
transdifferentiation in the ras-transformed HD6-4 colon carcinoma cells used in this study. ras transformation of
colon carcinoma cells disrupts their basolateral polarity and decreases their lateral adherence by down-regulation of N-cadherin (36). The
cells become rounded and less differentiated, are less adherent to
laminin and collagen I because of blocked maturation of integrin
1,
form multicellular aggregates, and grow much more rapidly in athymic
mice (35). TGF-
initiates several parallel signaling pathways with
cellular responses determined by the set of transcription factors that
are activated by separate and interacting TGF-
pathways (79). After
SMAD signaling is inhibited by mutant Ki-Ras, the TGF-
1 pathways
left functioning may interact with pathways activated by mutant Ki-Ras
to increase tumorigenicity. Such oncogenic ras-activated pathways include the post-translational modification of T
RIII described in this study, the post-translational modification of
1 integrin (35), increases in growth rate through
constitutive activation of MAPKs (35), and decreases in intercellular
adhesion through up-regulation of carcinoembryonic antigen
(36).