From the Departments of Medicine and
§ Biological Chemistry, The Johns Hopkins University School
of Medicine, Baltimore, Maryland 21205
Received for publication, December 11, 2000
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ABSTRACT |
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Intestinal-enriched Krüppel-like
factor (IKLF or KLF5) belongs to the family of mammalian
Krüppel-like transcription factors. Previous studies indicate
that expression of IKLF is enriched in the proliferating
crypt epithelial cells of the intestinal tract. However, the biological
function of IKLF is unknown. In the current study, we have shown that
the level of IKLF mRNA was nearly undetectable in
serum-deprived NIH3T3 fibroblasts but became acutely and significantly
increased upon the addition of fetal bovine serum or the phorbol ester,
PMA. This induction required protein synthesis because it was prevented
by cycloheximide. Transfection of IKLF into NIH3T3 cells
resulted in the formation of foci in a manner similar to that caused by
the activated Ha-ras oncogene. Constitutive
expression of IKLF in transfected NIH3T3 cells
significantly increased the rate of proliferation when compared with
cells transfected with an empty vector. The growth of
IKLF-transfected cells was no longer inhibited by cell-cell
contact or by low serum content. Moreover, these cells proliferated in
an anchorage-independent fashion. We conclude that
IKLF encodes a delayed early response gene product that
positively regulates cellular proliferation and may give rise to a
transformed phenotype when overexpressed.
Krüppel is a zinc finger-containing transcription
factor that is responsible for segmentation of the Drosophila
melanogaster embryo (1). In vertebrates, a large number of
proteins have been identified that exhibit homology to Krüppel
(2). One prominent example is Sp1 (3), a general transcription factor. Recently, a family of Krüppel-like factors
(KLFs)1 that are highly
related to the mammalian Krüppel protein erythroid Krüppel-like factor (EKLF) (4) have been described (2, 5, 6).
Many of these proteins were given a numerical designation by the Human
Gene Nomenclature Committee (HGNC, Ref. 7), with EKLF designated as
KLF1. The genes encoding many KLFs are expressed in a tissue-specific
or -selective manner. In addition, evidence suggests that KLFs
collectively exert important regulatory functions in diverse biological
processes such as growth, development, differentiation, and apoptosis.
One tissue in which a number of KLFs appear to play an important
regulatory role is the intestinal epithelium. This tissue is a dynamic
system in which proliferation of stem cells located in the crypts is
intimately coupled to their differentiation into mature daughter cells
once they exit the crypts (8, 9). Expression of the genes encoding two
KLFs, gut-enriched Krüppel-like factor (GKLF or KLF4, Ref. 10,
11) and intestinal-enriched Krüppel-like factor (IKLF or KLF5,
Refs. 12, 13), is particularly active in the intestinal epithelium.
However, their patterns of expression appear to be complementary rather
than redundant. Whereas GKLF is primarily expressed in the
differentiated epithelial cells, away from the proliferating zone,
IKLF is found mainly in the proliferating crypt cell
population. The in vivo pattern of GKLF expression is mirrored by its in vitro pattern; it is found
mostly in cells that are growth-arrested (10, 14). Moreover,
constitutive expression of GKLF leads to the inhibition of
DNA synthesis (10, 15). In contrast, the physiological function of IKLF
is less clear, although it has been proposed to have an opposing effect to GKLF in regulating epithelial cell differentiation (12).
The present study seeks to characterize the effect of IKLF on
cellular proliferation. We demonstrate that expression of
IKLF in cultured cells responds transiently and acutely to
growth stimuli. In addition, forced expression of IKLF in
transfected cells results in an accelerated rate of proliferation and a
transformed phenotype as evidenced by formation of foci, loss of
contact inhibition, as well as acquisition of serum- and
anchorage-independent growth. Our results indicate that IKLF has a
proproliferative effect, which lends support to the previous hypothesis
that it may counteract the function of GKLF.
Reagents--
Cell culture media and fetal bovine serum
(FBS) were purchased from Life Technologies (Gaithersburg, MD) and
Hyclone Laboratories (Logan, UT), respectively. Radioisotopes were
purchased from PerkinElmer Life Sciences. Phorbol 12-myristate
13-acetate (PMA) and cycloheximide (CHX) were purchased from Sigma. The
monoclonal antibody directed against the hemagglutinin A (HA) epitope
(F-7, sc-7392) was purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). Expression constructs containing full-length IKLF and
HA-tagged IKLF (pBK-CMV-IKLF and pBK-CMV-IKLF·HA, respectively) were
kindly provided by Dr. Jerry Lingrel (12). The expression construct
containing activated Ha-Ras was a generous gift of Dr. Raul Urrutia
(16). The expression construct containing full-length GKLF, PMT3-GKLF,
was previously described (10).
Cell Culture--
NIH3T3 cells were maintained in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 10% FBS, 100 µg/ml
streptomycin, and 100 units/ml penicillin at 37 °C in a 5%
CO2 atmosphere. For experiments involving mitogenic
stimulation, cells were first rendered quiescent by removal of serum
from the medium for 24 h. Cells were then stimulated with fresh
medium containing 15% FBS or 100 ng/ml PMA for various duration. In
experiments in which CHX was included, quiescent cells were pretreated
with 10 µg/ml CHX for 1 h before the addition of FBS or PMA.
Treatments were then continued for another 1 h with the respective
mitogen and CHX.
Northern and Western Blot Analyses--
RNA was isolated using
the Trizol method (Life Technologies) and resolved by denaturing
agarose gel electrophoresis followed by transfer to nylon membranes.
Complementary DNA probes encoding IKLF and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were labeled using [ Focus Formation Assay--
Focus formation assays were performed
according to a previously published protocol (17). Briefly, 5 µg of
plasmid DNA containing the various effectors was transfected into
NIH3T3 cells using LipofectAMINE (Life Technologies, Inc.). Cells were
then maintained in DMEM and 10% FBS for 2-3 weeks at which time they
were stained with methylene blue to demonstrate the foci. The number of
foci in each dish was then manually counted.
Establishment of Stable IKLF-expressing Cell Lines--
NIH3T3
cells were transfected with pBK-CMV-IKLF·HA or the pBK-CMV empty
vector using LipofectAMINE. Two mg/ml G418 was added to the medium
beginning 24 h after transfection to select for resistant clones,
which were isolated 2 weeks later and expanded. The presence of
HA-tagged IKLF was detected by Western blot analysis using the anti-HA
monoclonal antibody.
Cell Proliferation and Serum and Anchorage Dependence
Assays--
Cell proliferation assays were performed by seeding the
IKLF·HA- or empty vector-transfected cells at a
density of 2 × 105 cells per well in 6-well plates.
Cells were fed DMEM with 10% FBS every other day, and the number of
cells in the wells were manually counted with a hemocytometer daily for
up to 5 days following seeding. For the serum dependence assay, cells
were seeded at a density 2 × 105 cells per well in
6-well plates and maintained in DMEM with 1% FBS. Cells were counted
every other day up to 6 days after seeding. Anchorage dependence assay
was performed according to a published protocol (17).
Ha-ras-, IKLF·HA-, or empty
vector-transfected cells were seeded at a density of 5 × 105 cells per 10-cm dish in a 0.33% top agar suspension,
which was overlaid onto a 0.5% agar bottom layer. Cells were fed DMEM,
10% FBS, and 2 mg/ml G418 every other day. Colonies developed in the agar suspension were examined 3 weeks following seeding, and the number
was tabulated under an inverted phase-contrast microscope. Photodocumentation was accomplished with a Nikon digital camera.
IKLF Encodes a Delayed Early Response Gene to Growth
Stimulation--
To investigate the responsiveness of IKLF
expression to growth stimulation, we conducted Northern blot analyses
in cultured fibroblasts. NIH3T3 cells were first rendered quiescent by
removing the serum from the medium and then stimulated by 15% FBS or
the phorbol ester, PMA. As seen in Fig.
1, A and B, the
level of IKLF transcripts was barely detectable in
serum-starved, quiescent cells (time 0). Upon addition of
FBS (Fig. 1A) or PMA (Fig. 1B), the levels of
IKLF transcripts increased acutely and transiently, reaching
a maximum after 2-3 h of treatment before returning to baseline
levels. The enhancing effect of both stimuli was prevented in cells
pretreated with the protein synthesis inhibitor, cycloheximide (Fig.
1C, CHX). These results indicate that IKLF is an
early response gene to growth stimulation although this responsiveness
requires protein synthesis. IKLF would therefore fall into
the category of delayed early response genes (18, 19).
Forced Expression of IKLF Causes Formation of Foci--
As a means
to measure the effect of IKLF on cell proliferation, we conducted the
focus formation assay previously developed for testing the transforming
activity of oncogenes such as ras (17, 20). NIH3T3
fibroblasts were transfected with the various effector constructs and
foci scored 2-3 weeks after transfection. As shown in Fig.
2, an expression plasmid containing the
HA-tagged IKLF produced ~30% of the number of foci caused by
activated Ha-Ras. A second construct containing IKLF without the HA-tag
gave rise to similar results (not shown). In contrast, an expression
plasmid containing GKLF failed to produce any foci, as were
mock-transfected cells (Fig. 2). These results suggest that forced
expression of IKLF causes focus formation in a manner
similar to activated Ha-Ras, an activity that was not paralleled by
GKLF.
Constitutive Expression of IKLF Causes Accelerated Cell
Growth--
To further investigate the effect of IKLF on cell
proliferation, we established several clonally derived NIH3T3 cell
lines that had been transfected with pBK-CMV-IKLF·HA or the pBK-CMV empty vector and selected with the antibiotic, G418. Two independent clones from each construct were chosen and examined. Fig.
3A shows that the two
pBK-CMV-IKLF·HA- but not the two pBK-CMV-transfected clones
(lanes 1 and 2 versus lanes 3 and 4,
respectively) produced a full-length HA-tagged IKLF as detected by
Western blot using a monoclonal antibody against the HA epitope. When
the growth characteristics of these cells were examined and compared
over a course of 5 days following seeding at a low density, it became apparent that the IKLF·HA-expressing cells
proliferated at a much faster rate compared with the control, empty
vector-transfected cells (Fig. 3B). Moreover, whereas the
control cells ceased to proliferate after reaching confluency, the
IKLF·HA-transfected cells continued to grow to
several layers (Fig. 3C). These findings suggest that the
growth of IKLF·HA-transfected cells was no
longer subject to contact inhibition, providing further evidence for a
proproliferative effect of IKLF.
IKLF Causes Serum- and Anchorage-independent Growth--
The
stably transfected NIH3T3 cells were subjected to additional and more
stringent tests of cellular proliferation and transformation. In one,
cells were seeded and maintained in medium containing only 1% FBS.
Under these conditions, empty vector-transfected cells failed to
proliferate. In fact, many perished because of the low-serum content.
In contrast, IKLF·HA-transfected cells continued to proliferate at a relatively brisk rate albeit slightly slower than that when maintained in 10% FBS (compare Figs.
4A and 3B). A
second test involved growth in soft agar (17). As seen in Fig.
4B, IKLF·HA-transfected cells formed
colonies in an agar suspension as were Ha-ras-transfected
cells at a ~1:3 ratio. In contrast, empty vector-transfected cells
remained as single cells in the agar suspension without ever forming
any colonies (Fig. 4C). The morphology and size of the
colonies produced by IKLF·HA- and
Ha-ras-transfected cells were very similar to each other
(Fig. 4C).
Complementary DNA clones encoding mouse IKLF were initially
identified because of sequence homology to LKLF (12). A human homolog
was subsequently isolated based on its binding to a specific cis-sequence in the promoter of the lactoferrin gene
promoter (21). In situ hybridization studies of both adult
(12) and fetal intestinal tissues (13) showed that expression of
IKLF is concentrated in the base of intestinal crypts. The
in vivo pattern of IKLF expression in the
intestinal tract therefore correlates with a proliferative phenotype,
although a direct effect of IKLF on cellular proliferation was not
established by these studies. It should be noted that IKLF is identical
to the previously isolated BTEB2 (22), which, because of a sequencing
error, has a shorter open-reading frame than IKLF (13). In a rabbit
model, expression of BTEB2/IKLF is induced in
activated smooth muscle cells (SMCs) in the neointima of
balloon-injured aorta (23). Similarly, increased BTEB2
expression has been noted in proliferating SMCs at anastomotic vascular
stricture (24) and this increased expression is a positive predictive
factor for vascular restenosis in pathological conditions (25). Taken
together, these studies suggest that activation of
BTEB2/IKLF expression is correlated with a proliferative state.
The results of the current study indicate that expression of
IKLF in cultured, quiescent NIH3T3 cells is acutely and
transiently induced upon mitogenic stimulation by factors such as serum
and phorbol ester. This induction is dependent on new protein synthesis as it is abolished in the presence of cycloheximide. A similar inductive response of BTEB2/IKLF was noted in cultured
rabbit aorta-derived SMCs treated with PMA or basic fibroblast growth factor (26). The latter study also showed that expression of the
immediate early response gene, Egr-1, is highly up-regulated by PMA and that Egr-1 binds to and activates the promoter of the BTEB2/IKLF gene (26). It is therefore possible that Egr-1 is an immediate mediator of induction of IKLF during
proliferative responses.
Despite evidence from in vitro and in vivo
studies demonstrating a correlation between IKLF expression
and proliferation, it is not clear whether IKLF directly regulates
cellular proliferation. The results of the current study are the first
to show that constitutive expression of IKLF increases
proliferation. We also show that IKLF alone is sufficient to cause a
transformed phenotype as assessed by focus formation (Fig. 2), loss of
contact inhibition (Fig. 3C), and gain of serum- (Fig.
4A) and anchorage-independent growth (Fig. 4, B
and C). These observations therefore suggest that IKLF is
potentially a mediator of cellular proliferation in the various in vivo and in vitro systems described above.
Whether IKLF directly participates in regulating the cell cycle or
whether expression of IKLF is increased in pathological
conditions such as neoplasm remains to be investigated.
Based on the opposing patterns of expression of GKLF and
IKLF in the intestine, Lingrel and co-workers (12) proposed
that their gene products may have opposing effects in regulating
proliferation and differentiation of the intestinal epithelium. Indeed,
the antiproliferative effect of GKLF depicted by previous studies (10,
14) and the proproliferative effect of IKLF demonstrated by this study
support their hypothesis. Biochemical evidence also suggests that the
two proteins may have opposing functions. For example, the promoter of
the SMC differentiation marker gene, SM22
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
COMPLEMENTARY DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
COMPLEMENTARY DISCUSSION
REFERENCES
-32P]dATP and the random-primed DNA
labeling kit (Roche Molecular Biochemicals). Conditions of
hybridization and washing were previously described (10). Western blot
analysis was performed according to a previous protocol (10) using
proteins extracted from stably transfected cells. The blots were probed
with a monoclonal antibody directed against the HA epitope at a
concentration of 200 ng/ml. Blots were then incubated with a
peroxidase-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology) at
a dilution of 1:2,000. Signals were detected using enhanced
chemiluminescence (Amersham Pharmacia Biotech).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
COMPLEMENTARY DISCUSSION
REFERENCES
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Fig. 1.
Activation of IKLF expression in NIH3T3 cells
upon mitogenic stimulation. Before addition of mitogens, cells
were incubated in medium stripped of serum for 24 h. Medium
containing fresh FBS (15% v/v, A) or PMA (100 ng/ml,
B) were then added to the cells for the various times
indicated. RNA was extracted from the cells and probed simultaneously
for the IKLF and GAPDH transcript content. In
C, serum-deprived cells were preincubated in the absence
(lanes 1, 3, and 5) or presence
(lanes 2, 4, and 6) of 10 µg/ml CHX
for 1 h, followed by an additional 1 h of medium alone
(lanes 1 and 2), medium containing FBS
(lanes 3 and 4), or PMA (lanes 5 and
6) in the absence or presence of CHX. RNA was extracted and
probed for both IKLF and GAPDH.
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Fig. 2.
Focus formation assay. Focus formation
assay was conducted as described under "Experimental Procedures."
NIH3T3 cells were transfected with 5 µg/10-cm dish of an expression
plasmid containing Ha-Ras (16), IKLF·HA (12), or GKLF (10). The
control was mock-transfected NIH3T3 cells. Foci were visualized by
staining with methylene blue 2-3 weeks following transfection. Shown
are the means of four independent experiments, each conducted in
quadruplicate. Bars indicate S.E. *, p < 0.01 when compared with control, mock-transfected cells.
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Fig. 3.
Growth characteristics of cells stably
transfected with IKLF. Clonal derivatives of NIH3T3
cells stably transfected with pBK-CMV-IKLF·HA or pBK-CMV were
selected with G418 and analyzed for the content of IKLF·HA by Western
blot analysis using an anti-HA monoclonal antibody (A).
Shown are the results from two experiments each of
IKLF·HA- and empty vector-transfected clones
(I3 and I10 versus V3 and
V5, respectively). In B, cells from each clone
were seeded at a density of 2 × 105 cells/well in
6-well plates and fed every other day with DMEM containing 10% FBS.
Three wells of cells from each clone were counted daily in triplicate,
after seeding for up to 5 days. Shown are the mean cell numbers/well in
log scale of IKLF·HA- and empty
vector-transfected cells (I3 and I10
versus V3 and V5, respectively).
Bars indicate S.E. C, typical morphology of
IKLF·HA- and empty vector-transfected cells
(left versus right, respectively) at 5 days after seeding.
As shown, while the vector-transfected cells remained a monolayer, the
IKLF·HA-transfected cells grew to multiple
layers.
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Fig. 4.
Serum and anchorage dependence assays of
IKLF-transfected cells. A, cells from the
two IKLF·HA-transfected clones (I3
and I10) were seeded at a density of 2 × 105 cells/well in 6-well plates and maintained in 1% FBS
for up to 6 days after seeding. Cells were counted every other day as
described in the legend to Fig. 3 and plotted over time. The two empty
vector-transfected clones (V3 and V5) failed to grow (not shown).
B, Ha-ras-, IKLF·HA-, and
empty vector-transfected cells were seeded in agar suspension, which
were fed DMEM with 10% FBS every other day for 3 weeks. Colonies of
cells were scored by visual inspection using an inverted phase-contrast
microscope. Shown are the mean colony numbers in 8 random fields under
a × 100 magnification. Bars indicate S.E. *,
p < 0.005 compared with empty vector-transfected cells
(V3 and V5). C, photomicrograph of a typical colony of
Ha-ras- and IKLF·HA-transfected
cells (left versus middle, respectively). Empty
vector-transfected cells (right) produced no such
colonies.
COMPLEMENTARY DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
COMPLEMENTARY DISCUSSION
REFERENCES
, is repressed
by GKLF but activated by IKLF (27). Preliminary studies from our
laboratory also indicate that GKLF and IKLF regulate the
GKLF promoter in an opposing
manner.2 In view of the
highly conserved sequence in the zinc finger region of the two proteins
and the similar DNA sequences to which they bind (10, 11, 12, 14, 21),
it would not be surprising that the two KLFs may coordinately regulate
the expression of a group of genes through similar if not identical
cis-elements. Further studies will demonstrate the
biochemical mechanisms by which GKLF and IKLF antagonize each other in
the context of regulating complex biological processes such as cellular
proliferation and differentiation.
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ACKNOWLEDGEMENTS |
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We thank M. Conkright and Dr. J. Lingrel for providing pBK-CMV-IKLF·HA and pBK-CMV-IKLF (12) and Dr. R. Urrutia for providing the activated Ha-Ras (16) expression construct.
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FOOTNOTES |
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* This work was supported in part by Grants DK52230 and CA84197 from the National Institutes of Health and the John G. Rangos Charitable Foundation.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.
¶ To whom correspondence should be addressed: Dept. of Medicine, Ross 918, The Johns Hopkins University School of Medicine, 720 Rutland Ave., Baltimore, MD 21205. Tel.: 410-955-9691; Fax: 410-955-9677; E-mail: vyang@jhmi.edu.
Published, JBC Papers in Press, January 10, 2000, DOI 10.1074/jbc.C000870200
2 C. Mahatan and V. W. Yang, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: KLF, Krüppel-like factor; GKLF, gut-enriched KLF; BTEB2, basic transcription element binding protein 2; CHX, cycloheximide; DMEM, Dulbecco's modified Eagle's medium; Egr-1, early growth response gene-1; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, hemagglutinin; IKLF, intestinal-enriched Krüppel-like factor; PMA, phorbol 12-myristate 13-acetate; SMC, smooth muscle cell.
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