(Received for publication, November 28, 1994; and in revised form, January 9, 1995)
From the
-1,6-N-acetylglucosaminyltransferase V (GnT-V) (EC
2.4.1.155) that catalyzes
-1,6 branching in asparagine-linked
oligosaccharides is activated on viral or oncogenic transformation and
is associated with tumor metastasis. To study the molecular mechanisms
involved in regulation of expression of the GnT-V gene, we cloned cDNA
and genomic DNA for the enzyme (Saito, H., Nishikawa, A., Gu, J.,
Ihara, Y., Soejima, Y., Sekiya, C., Niikawa, N., and Taniguchi,
N.(1994) Biochem. Biophys. Res. Commun. 198, 318-327).
We found that transforming growth factor
(TGF
) specifically
induced GnT-V expression in mouse melanoma cells. The activity of GnT-V
was increased 24 h after the addition of TGF
and remained at high
levels up to 72 h. Northern blot analysis showed that the mRNA levels
of GnT-V were consistent with the increased activity. To further
investigate the nature of the induction, mRNA stability and
transcriptional activity were assayed. The enhancement of the GnT-V
mRNA expression resulted from prolonged mRNA stability, not from
increased transcription. Consequently, elevated mRNA levels were
observed even 72 h after the addition of TGF
. Lectin blot analysis
involving leukoagglutinin showed newly synthesized
-1,6 branching
structures in the sugar chains of a protein of approximately 130 kDa at
48 h after TGF
treatment. These results suggested that TGF
caused changes in the sugar chains of proteins in melanoma cells by
up-regulating GnT-V expression.
UDP-GlcNAc, -mannoside
-1,6-N-acetylglucosaminyltransferase V (GnT-V) (
)yields
-1,6-linked branching in complex-type,
asparagine-linked oligosaccharides, as shown in Fig. S1(1, 2) . GnT-V is an interesting
glycosyltransferase because some malignant phenotypes are associated
with changes in its activity. For example, the transformation of baby
hamster kidney cells caused by a tumor virus results in increases in
GnT-V activity and alteration of cell-surface
glycosylation(3, 4) . Recently, GnT-V was purified
from rat kidney (5) and human lung cancer cells(6) ,
and cloning of its cDNA and chromosomal mapping were
achieved(7, 8) . We reported (9) that
expression of GnT-V was induced in the early stages of
hepatocarcinogenesis in rodent models and that the increased levels of
GnT-V activity were consistent with the increased mRNA levels.
Scheme 1: Scheme 1.
Transforming growth factor 1 (TGF
) is widely referred to
as a prototypical epithelial cell inhibitor(10, 11) .
Although it inhibits the growth of both normal melanocytes and melanoma
cells(12, 13) , TGF
is expressed at higher levels
in many cancers, including melanomas, than in normal
tissue(14, 15, 16) . TGF
also exhibits a
number of properties that can promote tumor growth. It has been found
to stimulate angiogenesis(10) , to suppress the immune
system(17, 18) , and to modulate cellular adhesion and
migration on an extracellular matrix(19, 20) . The
expression of TGF
by cancer cells is thought to be related to
their malignant phenotype(20, 21) .
The structures
of sugar chains have a lot of biological functions, including cell
differentiation, cell adhesion, viral infection, and the action of
hormones(22) . Dennis et al.(23, 24) reported that high GnT-V activity and
-1,6 branching structures of sugar chains were directly associated
with metastasis. The molecular mechanism by which the GnT-V activity
increases is, however, unknown. In this study, we found that the
expression of GnT-V was induced specifically by TGF
in some
cytokines and found that
-1,6 branching structures in the sugar
chains of proteins increased after TGF
treatment. The mechanism
underlying the enhancement of GnT-V was investigated at the RNA level.
Nylon membranes (Zeta-Probe, Bio-Rad) were
prepared with immobilized GnT-V cDNA as described below. Bluescript II
KS (Stratagene) containing GnT-V cDNA (9) was
linearized with restriction enzymes and then precipitated with ethanol.
The linearized plasmid was denatured in 0.5 M NaOH and 2 M NaCl for 1 h at room temperature, neutralized with 6
SSC,
and then immediately blotted onto a nylon membrane using a slot-blot
apparatus (filtration manifold system, series 1055, Life Technologies,
Inc.). The membrane was hybridized with the radiolabeled RNA and then
washed as described for Northern blot hybridization. After development,
the intensities of the labeled bands were measured with a densitometer
(CS-9000, Shimadzu, Japan).
Figure 1:
Expression of GnT-V mRNA after
incubation with various reagents. F1 and F10 cells were cultured in the
presence of interleukin (IL) 1 (5 ng/ml), interleukin 2
(250 units/ml), TGF
(5 ng/ml), or hepatocyte growth factor (5
ng/ml) or in the absence of these reagents (control). After 48 h of
incubation with these reagents, total RNAs were extracted, and Northern
blot analysis was performed as described under ``Materials and
Methods.'' HGF, hepatocyte growth
factor.
Figure 2:
Changes in cell count after TGF
treatment. The number of cells was counted after incubation with
(
) or without (
) TGF
(5 ng/ml) for the times indicated.
Each point is the average of three values. Each bar indicates the standard deviation at the
point.
Figure 3:
Morphological changes in F1 and F10 cells
after TGF treatment. F1 and F10 cells were cultured in the
presence or absence of TGF
(5 ng/ml) for 24 h and then
photographed (original magnification,
200
).
Figure 4:
Effect of TGF on GnT-V activity in F1
cells. Cells were cultured in the presence (
) or absence (
)
of TGF
(5 ng/ml). GnT-V activity was assayed as described under
``Materials and Methods.'' Each point is the average
for triplicate experiments. Each bar indicates the standard
deviation at the point.
The effects of various
concentrations of TGF on GnT-V activity were investigated in both
F1 and F10 cells (Fig. 5). GnT-V activity increased in
proportion to the concentration of TGF
up to 2 ng/ml for F10 cells
and 5 ng/ml for F1 cells. The activity in F10 cells decreased at
TGF
concentrations of more than 5 ng/ml. In F10 cells, GnT-V
activity was induced at lower concentrations of TGF
than in F1
cells, although the initial levels were not different.
Figure 5:
Changes in GnT-V activity in F1 and F10
cells after incubation with various concentrations of TGF. F1
(
) and F10 (
) cells were cultured in the presence of various
concentrations of TGF
for 48 h. GnT-V activity was assayed as
described under ``Materials and Methods.'' Each point is the average for triplicate experiments. Each bar indicates the standard deviation at the
point.
Figure 6:
Effect of TGF on expression of GnT-V
mRNA. F1 cells were cultured in the absence (A, C, E) or presence (B, D, F) of
TGF
(5 ng/ml). Total RNA (30 µg) extracted from the cells at
the indicated times was electrophoresed on a 1.0% agarose gel
containing 2.2 M formaldehyde. The membrane filter was
hybridized with
P-labeled GnT-V cDNA (A, B) or
-actin (C, D). Ethidium bromide
staining shows a comparable amount of RNA in each lane (E and F).
Figure 7:
Effect of TGF on the stability of
GnT-V mRNA. F1 cells were cultured in the presence or absence of
TGF
(5 ng/ml) for 48 h. Total RNA was isolated from one set of
control and TGF
-treated cultures and used to determine the GnT-V
mRNA level at t = 0. The remaining cultures were placed
in fresh medium containing 100 µM DRB with or without
TGF
(5 ng/ml), followed by incubation for an additional 3, 4.5,
7.5, 12, or 18 h. Total RNA (30 µg) was isolated and analyzed for
GnT-V mRNA by Northern blot hybridization.
Figure 8:
Effect of TGF on transcription of
the GnT-V gene in nuclei from F1 cells. Nuclei were isolated from
control cells and cells treated with TGF
for 0-72 h. After
labeling with [
P]UTP, nascent RNA was purified
and hybridized to slots containing various concentrations of
immobilized Bluescript DNA containing GnT-V cDNA.
-Actin and
Bluescript (BS) DNA were used as positive and negative
control, respectively. The details of the assay procedure are given
under ``Materials and Methods.'' The relative intensities of
the labeled bands, GnT-V (
) and
-actin (
)
transcriptional levels in control F1 cells, and GnT-V (
) and
-actin (
) transcriptional levels in TGF
-treated cells
were measured, respectively, with a densitometer (A). Data
indicate the results for 1-3 bands. Representative results of
these experiments are shown in B.
Figure 9:
SDS-polyacrylamide gel electrophoresis
analysis and lectin blot analysis of F1 cells treated with TGF. A, 10 µg of total cellular proteins from homogenized F1
cells at 48 h (lane1), 72 h (lane2), and 96 h (lane3) without any
treatment or at 48 h (lane4), 72 h (lane5), and 96 h (lane6) after TGF
treatment were electrophoresed on a 12% agarose gel and then stained. B, 3 µg of the total cellular proteins mentioned above
were blotted onto a nitrocellulose membrane, and then staining with
L-PHA was performed as described under ``Materials and
Methods.'' The numbers at the left indicate the
molecular weights of standards.
TGF is a pleotrophic peptide that was originally shown
to induce a transformed phenotype in normal fibroblasts cultured in
soft agar(35) . It also regulates the growth of certain cell
types, primarily by inhibiting cell
proliferation(12, 13, 14, 15) .
Although the growth of F1 cells was inhibited by 18-33% on
incubation with TGF
(Fig. 2), GnT-V activity was
up-regulated by the TGF
treatment (Fig. 4). Therefore, the
effect of TGF
on the expression of GnT-V appears to be separate
from the function of the growth factor as a prototypical epithelial
cell inhibitor. Hahn and Goochee (36) reported that GnT-V
activity was higher in proliferating states and decreased in confluent
cultures. For this reason, the GnT-V activity in control F1 cells
slightly increased with time.
Melanoma cells differ from normal
melanocytes in morphology, life span, and chromosomal abnormality.
Morphology also differs between primary melanoma and metastatic
phenotypes(37) . Heffernan et al.(38) reported that -all-trans-retinoic
acid-induced endodermal differentiation of mouse teratocarcinoma cells,
F9, was accompanied by changes in the expression of
-1,6-N-acetylglucosamine transferase and polylactosamine.
Abrupt induction of GnT-V activity was observed in F9 cells after 4
days of retinoic acid treatment. Morphological changes accompanying
cell differentiation appeared at this time. While the GnT-V activity in
F1 cells gradually increased after incubation with TGF
and other
glycosyltransferases did not change ( Fig. 4and Table 1),
suggesting that different induction mechanisms are triggered by
TGF
and retinoic acid.
Melanoma cells produce a variety of
growth factors, including basic fibroblast growth factor,
platelet-derived growth factor, transforming growth factors and
(TGF
and TGF
), interleukins 1
and 1
, and
melanoma growth stimulatory activity(39) . The F1 and F10
cells, however, exhibited different sensitivities to exogenous TGF
(Fig. 5). The GnT-V activity in F10 cells increased at lower
concentrations of TGF
than that in F1 cells did. This might
reflect the high metastatic character of F10 cells. However, GnT-V
activities in steady states and L-PHA blotting pattern between F1 and
F10 cells were almost the same. The 130-kDa band as shown in Fig. 9B was induced by more than 2 ng/ml TGF
in
both cells (data not shown). No other differences except sensitivities
to exogenous TGF
were observed between F1 and F10 cells in the
present experiments.
TGF increased GnT-V activity by enhancing
mRNA expression (Fig. 6). The level of GnT-V mRNA increased
after 24 h of incubation with TGF
and remained high up to 72 h.
Because direct induction of GnT-V gene expression would likely follow a
shorter time course, a secondary effect of TGF
on GnT-V expression
was suggested. One possible mechanism involves increasing stability of
the mRNA. There is some evidence that up-regulation of extracellular
matrix synthesis by TGF
also affects post-transcriptional steps.
For example, TGF
treatment has been shown to increase the
half-lives of mRNAs for
1(I) collagen in confluent 3T3
fibroblasts(40) , heparan sulfate proteoglycan in colon cancer
cells(41) , and elastin in human skin fibroblasts(42) .
In contrast, up-regulation of collagen type II by TGF
in rabbit
articular chondrocytes (43) and glucocorticoids in normal human
T lymphocytes (44) involves increased transcription of the
genes without apparent changes in mRNA stability. TGF
appears to
up-regulate the GnT-V gene through the former mechanism ( Fig. 7and 8). The half-life of GnT-V mRNA increased
approximately 2.3-fold after TGF
treatment. The persistence of an
elevated level of GnT-V mRNA even after 72 h incubation with TGF
(Fig. 6) is in keeping with this mechanism. Although high
activities of GnT-V are observed in transformation and
carcinogenesis(3, 4, 9) , the mechanism
underlying the high expression remains to be elucidated. The induction
of GnT-V by TGF
is one possible pathway. The enhancement of GnT-V
expression by TGF
resembled that of some extracellular matrix
proteins induced by TGF
(40, 41, 42) .
Such regulation might be of significance as to characteristics of
GnT-V, a glycosyltransferase associated with tumor metastasis.