![]()
|
|
||||||||
(Received for publication, December 29, 1995, and in revised form, April 16, 1996)
§,
''
From the Departments of
Medicine and
Biological Chemistry, The Johns Hopkins University School of
Medicine, Baltimore, Maryland 21205 and the ¶ Center for Molecular
Medicine, Institute of Biotechnology, University of Texas Health
Science Center, San Antonio, Texas 78250
A cDNA clone, named gut-enriched Krüppel-like factor (GKLF), was isolated from an NIH 3T3 library using a probe encoding the zinc finger region of the immediate-early transcription factor zif/268. The deduced GKLF amino acid sequence contains three tandem zinc fingers that are related to members of the Krüppel family of transcription factors. By indirect immunofluorescence, GKLF is localized to the cell nucleus. In cultured fibroblasts, GKLF mRNA is found in high levels in growth-arrested cells and is nearly undetectable in cells that are in the exponential phase of proliferation. The growth-arresting nature of GKLF is demonstrated by an inhibition of DNA synthesis in cells transfected with a GKLF-expressing plasmid construct. In the mouse, GKLF mRNA is present in select tissues and is most abundant in the colon, followed by the testis, lung, and small intestine. In situ hybridization experiments indicate that GKLF mRNA is enriched in epithelial cells located in the middle to upper crypt region of the colonic mucosa. Taken together, these results suggest that GKLF is potentially a negative regulator of cell growth in tissues such as the gut mucosa, where cell proliferation is intimately coupled to growth arrest and differentiation.
Eukaryotic transcription factors are classified according to the
structural motif that contacts the DNA. The zinc finger motif is one
such example, in which a zinc atom is tetrahedrally coordinated by 4 amino acid residues (usually cysteine or histidine) within a 30-amino
acid sequence to form the DNA-binding domain. The biological importance
of this structure is reflected by estimates that the human genome has
between 300 and 700 genes containing the zinc finger motif (Klug and
Schwabe, 1995
; Hoovers et al., 1992
). A subset of the zinc
finger transcription factors contain amino acid sequences that resemble
those of the Drosophila segmentation gene product
Krüppel (Schuh et al., 1986
). They are characterized
by multiple zinc fingers containing the conserved sequence
X2
X3FX5LX2
X3
(X is any amino acid, and the underlined cysteine and
histidine residues are involved in the coordination of zinc) that are
separated from each other by a highly conserved 7-amino acid
inter-finger spacer, TGEKP(Y/F)X, often referred to as the
H/C link. These Krüppel-like proteins are involved in diverse
aspects of eukaryotic gene regulation such as cell growth and/or
differentiation (e.g. Egr-1 or zif/268 (Christy et
al., 1988
; Sukhatme et al., 1988
) and the erythroid
Krüppel-like factor (EKLF)1 (Miller
and Bieker, 1993
)), general transcription (e.g. Sp1
(Kadonaga et al., 1987
)), oncogenesis (e.g. WT1
(Wilms' tumor gene) (Call et al., 1990
) and Gli (involved
in gliomas) (Kinzler et al., 1988
)), and embryogenesis
(e.g. Krüppel (Schuh et al., 1986
) and
Hunchback (Stanojevic et al., 1989
)).
The eukaryotic cell cycle is a carefully controlled event that requires
the participation of several zinc finger-containing transcription
factors. It is divided into four major phases: G1, S,
G2, and M (Hartwell and Weinert, 1989
). Cells that do not
divide are considered quiescent and reside in a special niche called
the G0 or growth-arrested state. When quiescent cells are
given fresh medium containing serum, a cascade of cellular events occur
that culminate in DNA synthesis and subsequent cell division. Genes
that are induced shortly after quiescent cells are stimulated by serum
or purified growth factors are often referred to as the
``immediate-early'' genes. Among these are several that encode zinc
finger-containing transcription factors such as the Egr family of
proteins (Sukhatme, 1992
) (which includes zif/268 (Lau and Nathans,
1987
) and nup475 (DuBois et al., 1990
)). The functions of
many of these gene products are thought to induce the expression of
subsequent ``delayed-early'' genes that are directly involved in DNA
synthesis (Lanahan et al., 1992
).
A different facet of eukaryotic cell cycle regulation is manifested by
proteins that exert negative impacts on cell growth. The tumor
suppressor p53, for example, plays a prominent role in the arrest of
cell growth induced by DNA damage (Hartwell and Kastan, 1994
).
Recently, a group of proteins have been identified and shown to inhibit
the activities of the G1 cyclins and their associated
cyclin-dependent kinases, both of which are essential for
the orderly progression of the cell cycle. The mechanisms by which
these inhibitors act help explain how antiproliferative signals arrest
cells in the G1 phase in such diverse processes as the
repair of DNA damage, terminal differentiation, and cell senescence
(Sherr, 1993
). Thus, expression of one such inhibitor, p21, is induced
in response to DNA damage in a p53-dependent manner
(Hartwell and Kastan, 1994
). Similarly, the myogenic transcription
factor MyoD causes terminal withdrawal of cells from the cell cycle by
concomitantly inducing p21 expression and myocyte differentiation
(Halevy et al., 1995
). These findings suggest that depending
on the signaling event, the mammalian cell cycle is regulated by both
positive and negative control mechanisms.
In this study, we describe the identification and characterization of GKLF, a zinc finger-containing nuclear protein that is a member of the Krüppel family of transcription factors. Expression of GKLF in cultured fibroblasts is highest in growth-arrested cells and lowest in cells in the exponential phase of proliferation. Moreover, constitutive expression of GKLF in transfected cells results in the inhibition of DNA synthesis. In vivo, GKLF mRNA is very abundant in the crypt epithelium of the mouse colon. These findings suggest that GKLF may act as a negative regulator of proliferation of intestinal epithelial cells.
Restriction endonucleases and modifying enzymes
were purchased from New England Biolabs Inc. (Beverly, MA). Sequenase
was purchased from U. S. Biochemical Corp. Radioisotopes were
purchased from DuPont NEN. Media and serum were purchased from Life
Technologies, Inc. and Hyclone Laboratories (Logan, UT), respectively.
Fluorescein isothiocyanate-, Texas Red-, and horseradish
peroxidase-conjugated secondary antibodies were purchased from Amersham
Corp. The prokaryotic expression vectors pET3d and pET16b (Studier
et al., 1990
) were purchased from Novagen (Madison, WI). The
constitutive eukaryotic expression vector PMT3 (Swick et
al., 1992
), which utilizes the simian virus 40 enhancer and the
adenovirus major late promoter for expression, was obtained with
permission from the Genetics Institute (Cambridge, MA).
An NIH 3T3 cDNA
library in bacteriophage
gt10 (Lanahan et al., 1992
) was
kindly provided by Dr. Ty Lanahan (The Johns Hopkins University). This
library was screened under conditions of reduced stringency with a
radioactively labeled DNA fragment containing the zinc finger portion
of the immediate-early transcription factor zif/268 (Christy et
al., 1988
). One of the positive clones, A14, contained a DNA
insert of ~1900 base pairs (bp) and was selected for further
investigation as it appeared to encode a novel gene based on partial
sequence analysis. Radioactively labeled A14 cDNA was used to
rescreen the same library under high stringency conditions, which
yielded >50 positively hybridizing plaques. The plaque with the
longest DNA insert, ~2800 bp in length, was amplified, and its insert
was subcloned into the plasmid pBluescript (Stratagene, La Jolla, CA).
Overlapping restriction endonuclease fragments of the cDNA with
sizes of <400 bp were further subcloned and sequenced bidirectionally
by the dideoxy chain termination method using the Sequenase kit (U. S.
Biochemical Corp.). Sequence comparison was performed using the BLAST
algorithm provided by the National Center for Biotechnology Information
(Rockville, MD).
Complementary DNA containing the open reading frame (ORF)
of GKLF was subcloned into pET3d and pET16b to generate
pET3d-GKLF and pET16b-GKLF, respectively. The pET3d-GKLF construct was
introduced into the BL21(DE3) strain of Escherichia coli
cells, which were subsequently induced by the addition of 0.4 mM isopropyl-
-D-thiogalactopyranoside to
produce GKLF. The protein, present largely in the form of inclusion
bodies, was solubilized by the method of Fraser et al.
(1993)
and separated by SDS-polyacrylamide gel electrophoresis. GKLF
was electroeluted from the gel and used to raise a rabbit polyclonal
antiserum by HRP Inc. (Denver, PA).
Transient transfections were performed by
the DEAE-dextran technique (Lopata et al., 1984
) or by
lipofection (Felgner et al., 1987
) using the Lipofectin
reagent as recommended by the manufacturer (Life Technologies, Inc.).
The ORF of the GKLF cDNA was subcloned into the
constitutive mammalian expression vector PMT3 to generate PMT3-GKLF.
Transfections of the monkey kidney-derived cell line COS-1 (American
Type Culture Collection, Rockville, MD) were accomplished with 1 µg/ml PMT3-GKLF DNA in 3.5- or 10-cm culture dishes. Two days
following transfection, cells were examined for GKLF production by
Western blot or immunocytochemical analysis (see below).
For [3H]thymidine incorporation, proliferating COS-1 cells were transfected with 1 µg/ml PMT3-GKLF or control PMT3 DNA in 3.5-cm culture dishes. Twenty-four h following transfection, cells were incubated with 1 µCi/ml [3H]thymidine (20 Ci/mmol) at 37 °C for 3 h. After washing twice with cold phosphate-buffered saline (PBS), cells were fixed with 10% trichloroacetic acid at 4 °C for 30 min, rinsed with 10% trichloroacetic acid, solubilized with 1 N NaOH, and neutralized with HCl. Aliquots equal to 0.1 volume of the solubilized material were counted in triplicate by liquid scintillation. Dishes that contained no cells were labeled and counted the same way to provide background counts.
Western Blot AnalysisSDS-polyacrylamide gel
electrophoresis was performed according to Laemmli (1970)
with the
following modifications. The acrylamide concentrations of the stacking
and running gels were 5 and 10%, respectively. Protein samples were
dissolved in loading buffer (60 mM Tris-HCl, pH 6.8, 2%
SDS, 100 mM dithiothreitol, and 0.01% bromphenol blue);
heated to 100 °C for 3 min; and loaded onto the gel in
electrophoresis buffer containing 25 mM Tris-HCl, pH 8.3, 250 mM glycine, and 0.1% SDS. At the completion of
electrophoresis, proteins were transferred to nitrocellulose membranes
according to the method of Towbin et al. (1979)
and
immunoblotted with rabbit anti-GKLF serum (1:1000 dilution) or
preimmune serum. Following incubation with the secondary antibody
(horseradish peroxidase-conjugated donkey anti-rabbit IgG), GKLF was
visualized with enhanced chemiluminescence (Amersham Corp.).
Immunocytochemical studies of transiently transfected COS-1 cells grown on plastic coverslips were performed 2 days following transfection. Coverslips were washed with PBS and fixed in 3% paraformaldehyde in PBS for 20 min. Cells were then permeabilized with 0.1% Nonidet P-40 in PBS for 10 min, washed with PBS, blocked with 10% fetal calf serum at 37 °C for 15 min, and incubated with rabbit anti-GKLF serum (1:500 dilution) in PBS at room temperature for 1 h. After washing with PBS, the coverslips were incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (1:200 dilution) at room temperature for 1 h, mounted in 25% glycerol with 1 mg/ml phenylenediamine, and visualized with a Zeiss Axioskop 20 microscope equipped for epifluorescence. For bromodeoxyuridine (BrdUrd) labeling, cells were first fed DMEM with 0.5% FCS for 24 h after transfection, followed by DMEM with 10% FCS and 100 µM BrdUrd (Sigma) for an additional 24 h. Coverslips bearing transfected cells were washed with PBS, fixed with 3% paraformaldehyde in PBS, permeabilized with 0.25% Triton X-100 in PBS for 10 min, and washed with PBS followed by water. Coverslips were then treated with 2 N HCl at room temperature for 30 min to denature the DNA, neutralized with 0.1 M sodium borate at room temperature for 5 min, and washed with PBS. A mouse monoclonal antibody raised against BrdUrd (1:5 dilution; Sigma B-2531) was then added to the coverslips at 37 °C for 2 h. Following washing with PBS, rabbit anti-GKLF serum (1:2000 dilution) was added to the coverslips at room temperature for 15 min, after which they were washed with PBS. Coverslips were subsequently incubated with a mixture of fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (1:200) and Texas Red-conjugated sheep anti-mouse IgG (1:20) at 37 °C for 1 h, mounted, and visualized with a fluorescence microscope as described above.
RNA Isolation and Northern Blot AnalysisRNA was isolated
from NIH 3T3 cells and from various mouse tissues by the guanidinium
thiocyanate method (Chirgwin et al., 1979
). Twenty µg of
total RNA were size-fractionated in 1.2% agarose gels containing 2.4 M formaldehyde (Ausubel et al., 1991
) and
transblotted onto nylon membranes (Hybond-N, Amersham Corp.).
Hybridizations and washings were performed under high stringency
conditions as described by Oliva et al. (1993)
using a
radioactively labeled GKLF cDNA probe. To control for
loading of RNA samples, all blots were stripped and reprobed with a
radioactively labeled DNA fragment encoding the 18 S ribosomal RNA gene
and, in some cases, with a cDNA fragment encoding the
constitutively expressed glyceraldehyde-3-phosphate dehydrogenase gene
(CLONTECH, Palo Alto, CA). When RNA from fractionated colonic tissue
was used, the inner surface of an everted piece of freshly obtained
mouse colon was gently scraped with a razor blade, and RNA was isolated
as described above. This method yields a highly enriched population of
cells of epithelial origin as noted before (Oliva et al.,
1993
).
A 575-bp
PflMI-KpnI restriction endonuclease fragment of
the GKLF cDNA was subcloned into pBluescript, and the
recombinant plasmid was used to generate 35S-labeled
antisense or sense RNA probe by in vitro transcription using
T7 or T3 RNA polymerase, respectively. Freshly isolated mouse colon was
fixed in 10% buffered Formalin solution, embedded in paraffin,
sectioned into 5-µm slices, and layered onto microscope slides.
Slides were processed for in situ hybridization as described
previously (Tietjen et al., 1994a
, 1994b
) and probed with
either the antisense or control sense probe at high stringency. After
washing, slides were submerged in Kodak NTB-2 liquid emulsion and
stored in light-proof boxes. After development, slides were
counterstained with hematoxylin and eosin and viewed under dark-field
microscopy.
To identify transcription
factors that are involved in growth regulation, a cDNA library
generated with mRNA from NIH 3T3 cells that were rendered quiescent
and stimulated with serum for 3 h (Lanahan et al.,
1992
) was screened under reduced stringency conditions with a DNA probe
containing the zinc finger region of the immediate-early transcription
factor zif/268 (Christy et al., 1988
). Partial sequence
analysis of the cDNA insert of a positive clone, A14, showed that
it did not have any significant sequence identity to those stored in
the GenBankTM DNA data base. The 1.9-kilobase insert of the
A14 clone was used to rescreen the same library under high stringency
hybridizing and washing conditions, which resulted in the
identification of >50 positive clones. The clone with the longest
insert, which we later named GKLF, was chosen for further
analysis. Its complete nucleotide sequence was determined and is shown
in Fig. 1.
Fig. 1.
Complete nucleotide and deduced amino acid
sequences of the GKLF. Numbers on the left refer
to the amino acid sequence, and those on the right refer to the
nucleotide sequence. Diagrammed beneath the sequence is a schematic
presentation of the GKLF open reading frame. N, amino
terminus; C, carboxyl terminus. The hatched areas
represent the three zinc fingers (Zn). The three
shaded areas are (1) a putative sequence recognized by SH3
domain-containing proteins (SH3), (2), a PEST sequence
(PEST), and (3) a potential nuclear localization signal
(NLS). P and S depict the proline- and
serine-rich regions. The nucleotide and amino acid sequences have been
deposited in GenBankTM under the accession number
U20344[GenBank].
Analysis of GKLF cDNA and Deduced Amino Acid Sequences
The GKLF cDNA contains a 311-bp
5
-untranslated region, a single ORF of 1449 bp, and a 977-bp
3
-untranslated region that is trailed by a poly(A) tail. The ORF
potentially encodes a polypeptide of 483 amino acids with a predicted
molecular mass of 53 kDa. Three potential methionine initiation codons
are present in frame near the amino terminus (amino acids 1, 10, and
51), although nucleotide sequences surrounding the second methionine
codon conform more closely to Kozak's rule for translation initiation
(Kozak, 1987
). A consensus sequence for polyadenylation, AATAAA
(Proudfoot and Brownlee, 1976
), is found 24 bp upstream of the poly(A)
tail.
The deduced GKLF amino acid sequence contains three tandem zinc finger
motifs at the carboxyl terminus. The three zinc fingers are closely
related to the consensus sequence
CX2-4 CX3FX5LX2HX3H
for zinc finger-containing transcription factors and are separated from
each other by a 7-amino acid inter-finger spacer similar to the H/C
link consensus sequence, TGEKP(Y/F)X. These features
classify GKLF as a member of the Krüppel family of proteins
(Chowdhury et al., 1987
; Morris et al., 1994
;
Schuh et al., 1986
). GKLF is rich in proline and serine
residues, which constitute 12.8 and 11.6% of the total amino acids,
respectively. The clustering of these 2 amino acids is reminiscent of
the transactivation domains of previously established transcription
factors (Nakamura et al., 1993
). In addition, a potential
nuclear localization signal (Boulikas, 1993
) that is rich in arginine
and lysine residues is found between amino acid residues 384 and 390. A
``PEST'' sequence, which is found in proteins with intracellular
half-lives of <2 h (Rogers et al., 1986
), is present
between amino acid residues 113 and 152. Finally, a 7-amino acid
sequence, PPLPGRP, present between amino acid residues 55 and 61, is
highly related to the consensus sequence to which proteins containing
the SH3 domain bind (Alexandropoulos et al., 1995
; Ren
et al., 1993
). Taken together, these features suggest that
GKLF is a nuclear-localized transcription factor.
A search in the GenBankTM protein data base for amino acid
sequences related to that of GKLF revealed many sequences similar to
its zinc finger-containing region. In particular, the closest alignment
was found with three recently identified eukaryotic transcription
factors: 1) the lung Krüppel-like factor (LKLF) (Anderson
et al., 1995
); 2) EKLF (Miller and Bieker, 1993
); and 3)
basic transcription element-binding protein 2 (BTEB2) (Sogawa et
al., 1993
), expressed in the placenta and testis. The degree of
sequence identity in the 82-amino acid zinc finger region of GKLF to
these proteins is 92, 84, and 82%, respectively (Fig.
2). Other sequences that are related (but less
conserved) to the zinc finger region of GKLF include WT1 (Call et
al., 1990
) and Sp1 (Kadonaga et al., 1987
), showing 59 and 52% sequence identity, respectively. In contrast, the amino acid
sequence outside the zinc finger region of GKLF bears no significant
homology to any previously identified proteins with one notable
exception: the 20 amino acids immediately preceding the first cysteine
residue of the first zinc finger are 90% identical between GKLF and
LKLF (Anderson et al., 1995
). These findings indicate that
GKLF is a newly identified polypeptide.
When the nucleotide sequence of GKLF was compared with those
stored in the GenBankTM nucleic acid data base, two
additional cDNA clones with significant homology were identified.
Both sequences correspond to the extreme 3
-end of the 3
-untranslated
region of the GKLF cDNA. The first
(GenBankTM accession number D25944[GenBank]) was obtained during a
random cDNA sequencing analysis of expressed genes in human colonic
mucosa.2 It contains 330 bp of sequence
that exhibits 87% nucleotide identity to GKLF. The second
cDNA, named clone 59 (GenBankTM accession number
L26292[GenBank]), was obtained during a differential screening of a cDNA
library made from rat Sertoli cells that had been stimulated with
follicle-stimulating hormone (Hamil and Hall, 1994
). This cDNA is
800 bp long and is 93% identical to the GKLF sequence. The
high degree of sequence identity in the 3
-untranslated region between
GKLF and these two partial cDNA clones suggests that the
latter two potentially represent the human and rat homologues of
GKLF, respectively. It is of interest to note that in the
mouse, GKLF is expressed in the colon and testis (see
below).
To characterize the
protein encoded by GKLF, a cDNA fragment containing the
ORF was subcloned into two prokaryotic expression vectors, pET3d and
pET16b (Studier et al., 1990
), the latter creating a fusion
protein, which contains an additional 3 kDa of bacterial sequence when
expressed. The BL21(DE3) strain of host E. coli cells was
transformed with pET3d-GKLF and induced with
isopropyl-
-D-thiogalactopyranoside to produce GKLF. The
protein was resolved by SDS-polyacrylamide gel electrophoresis,
purified, and used to generate a rabbit polyclonal antiserum. The same
GKLF ORF cDNA fragment was also cloned into the
mammalian expression vector PMT3 (Swick et al., 1992
), and
the resultant construct was used to express GKLF in transiently
transfected COS-1 cells. Fig. 3A is an
immunoblot of proteins isolated from pET16b-GKLF-transformed E. coli and PMT3-GKLF-transfected COS-1 cells. A single prominent
polypeptide band with an apparent molecular mass of 56 kDa from the
transformed and
isopropyl-
-D-thiogalactopyranoside-induced bacteria was
detected by anti-GKLF serum (lane 1). Similarly, a
polypeptide band of 53 kDa was detected in lysates of COS-1 cells
transiently transfected with PMT3-GKLF (lane 2). The higher
apparent molecular mass of GKLF in lane 1 was due to the
presence of bacterial sequence in the recombinant protein. In contrast,
no antigens were detected by anti-GKLF serum in lysates of COS-1 cells
transfected with the PMT3 vector only (lane 3), indicating
that these cells contained little or no endogenous GKLF. The
specificity of the antiserum was demonstrated by the ability of
purified, bacterially produced GKLF to block the immunoreactivity of
the 53-kDa protein band in PMT3-GKLF-transfected COS-1 cells (data not
shown).
-D-thiogalactopyranoside and from COS-1 cells
transiently transfected with PMT3-GKLF (lane 2) or PMT3
(lane 3) using anti-GKLF serum. Molecular mass markers are
shown on the left. GKLF was seen as a single prominent band in both
lanes 1 and 2. The minor low molecular mass
protein bands noted were variable among different experiments and
probably represented degradation products. B, indirect
immunofluorescence of COS-1 cells transiently transfected with
PMT3-GKLF and immunostained with anti-GKLF serum. Six cells were noted
to express GKLF and exhibited intense nuclear staining. In contrast,
the nonexpressing cells showed only a background level of cytoplasmic
staining.
The intracellular localization of GKLF in mammalian cells was examined by indirect immunofluorescence analysis of COS-1 cells transiently transfected with PMT3-GKLF using anti-GKLF serum. As shown in Fig. 3B, GKLF was primarily localized to the cell nucleus in cells that expressed the transfected gene. In contrast, nonexpressing cells showed staining only at background levels (Fig. 3B), as did cells transfected with the PMT3 vector alone and immunostained under identical conditions (data not shown).
Expression of GKLF in Response to Serum Stimulation in Cultured FibroblastsGKLF was isolated from an NIH 3T3 cDNA
library generated with RNA from cells that had been rendered quiescent
and then stimulated for 3 h with serum. The serum responsiveness
of GKLF in NIH 3T3 cells was therefore examined by Northern
blot analysis. The mRNA content of GKLF from
growth-arrested quiescent NIH 3T3 cells (which had been maintained in
0.5% FCS for 5 days) was compared with that from cells induced to
enter the cell cycle by the addition of medium containing 15% FCS for
various lengths of time between 0 and 24 h. In addition, RNA
isolated from proliferating cells in the exponential phase of growth
was analyzed. As shown in Fig. 4, it is apparent that a
significant level of GKLF mRNA was present in quiescent
cells (lane 2), but was nearly absent in actively
proliferating cells (lane 1). The addition of 15% FCS not
only failed to increase the abundance of GKLF mRNA, but
a decrease in the message content was detected beginning at 8 h
after treatment. By 24 h after serum stimulation, a partial
recovery of the GKLF mRNA level was observed. Fig.
5 summarizes the results of quantitative densitometric
measurements of mRNA band intensities from Northern blot analyses
of three independent experiments. The data confirmed that the level of
GKLF transcript was much higher in growth-arrested cells
compared with proliferating cells and that when cells were induced to
enter the cell cycle, a reproducible decrease in the transcript level
was observed beginning at a time period that precedes the commencement
of DNA synthesis in NIH 3T3 cells (Quelle et al., 1993
).
The growth arrest-specific nature of GKLF expression was
confirmed by a reverse experiment to that of Fig. 4 in which NIH 3T3
cells in the exponential phase of proliferation were induced to enter
quiescence by a reduction of the serum content in the medium from 10 to
0.5% for various lengths of time. Fig. 6 shows that the
initial GKLF transcript was barely detectable, which only
became apparent beginning 2 days after the cells were deprived of
serum. A further increase in the GKLF mRNA level was
seen between days 2 and 3, after which nearly equivalent levels of
transcript were present up to 7 days. Similarly, when actively
proliferating NIH 3T3 cells were left in 10% FCS without any
additional feedings, levels of GKLF mRNA increased in a
time-dependent manner (Fig. 7). This result
was in clear contrast to the largely constant levels of the 18 S
ribosomal RNA and of the mRNA encoding glyceraldehyde-3-phosphate
dehydrogenase (Fig. 7). In this experiment, cell-to-cell contact seemed
to play a role in the increased expression of GKLF as cells
were fully confluent by day 2 of the experiment. Taking the results of
Figs. 6 and 7 together, it appears that expression of GKLF
is correlated with quiescence induced by serum deprivation and
withdrawal from the cell cycle due to contact inhibition.
Effect of Constitutive GKLF Expression on DNA Synthesis
The results of the preceding experiments suggest that expression of GKLF is temporally associated with growth arrest and that a down-regulation of GKLF occurs in cells that are stimulated to enter the cell cycle. To assess the effect of constitutive expression of GKLF on cell growth, COS-1 cells were transiently transfected with PMT3-GKLF or PMT3 for 24 h, at which time DNA synthesis was determined by [3H]thymidine incorporation. Table I shows the results of three independent experiments. In each experiment, the percentage of cells expressing GKLF in PMT3-GKLF-transfected cells was determined in parallel dishes by indirect immunofluorescence and found to vary between 25 and 30%. As shown, a statistically significant decrease in the incorporation of [3H]thymidine by cells transfected with PMT3-GKLF as compared with cells transfected with vector alone was observed in all three experiments. The close correlation between the degree of inhibition of [3H]thymidine incorporation into PMT3-GKLF-transfected cells and the percentage of cells expressing GKLF suggests that GKLF, when constitutively expressed, inhibits DNA synthesis.
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
To demonstrate directly that the cells expressing GKLF do not
synthesize DNA, PMT3-GKLF-transfected COS-1 cells were first incubated
with BrdUrd and then double-stained for GKLF and BrdUrd by indirect
immunofluorescence. As shown in Fig. 8, two cells that
expressed GKLF (arrowheads) failed to stain for BrdUrd. In
contrast, four non-GKLF-expressing cells were positive for BrdUrd.
After counting 100 consecutive GKLF-expressing cells, only five were
noted to positively stain for BrdUrd, whereas >80% of the
non-GKLF-expressing cells were BrdUrd-positive.
Tissue Distribution of GKLF
To determine the tissue
distribution of GKLF, RNA was isolated from various adult
mouse tissues and examined by Northern blot analysis. Of the tissues
examined, the colon (both proximal and distal) contained the highest
level of GKLF transcript (Fig. 9). Moderate
levels of transcript were also noted in the distal small intestine
(SI), testis, and lung. In addition, a small amount of
GKLF transcript was present in the proximal small intestine.
Smaller mRNA species were also noted in the intestinal tissues and
testis and could represent other closely related sequences or products
of alternative splicing. No appreciable amount of message was detected
in the brain, kidney, liver, spleen, thymus, heart, muscle, and fat.
These results indicate that the expression of GKLF is
tissue-selective and is most abundant in the colon.
The mouse colon is composed of a heterogeneous population of cells,
ranging from the epithelial cells lining the inner mucosal surface to
various other non-epithelial cell types such as lymphocytes,
fibroblasts, enteric neurons, and smooth muscle cells. To further
localize the cellular origin of GKLF, Northern blot analysis
was performed on RNA isolated from colonic mucosal scrapings (Fig.
10, lane 2), which represent an enrichment
of the epithelial cell population as previously demonstrated (Oliva
et al., 1993
). As a comparison, Northern blot analysis was
also performed on RNA isolated from the remaining colonic tissue after
scraping (Fig. 10, lane 3). As shown, GKLF
mRNA was highly enriched in the mucosal population of cells,
suggesting that GKLF is mainly expressed in epithelial
cells. The epithelium-specific expression of GKLF was
confirmed by in situ hybridization. Fig.
11A shows that the antisense GKLF
RNA probe hybridized primarily to the middle to upper region of the
colonic crypt epithelium. In contrast, the control sense
GKLF RNA probe produced a random background distribution of
silver grains (Fig. 11B). These results indicate that
GKLF is epithelium-specific and is expressed primarily in
cells that are in the process of migrating from the base toward the top
of the crypt.
The cDNA encoding GKLF was isolated by low stringency
hybridization to a zinc finger-containing transcription factor,
zif/268. Although not proven in this study, several features of GKLF
strongly suggest that it is a transcription factor. First, the amino
acid sequence at the carboxyl terminus containing the zinc fingers is
similar to those of a number of proteins, many of which are proven
transcription factors, such as LKLF, EKLF, and BTEB2 (Fig. 2). Second,
GKLF contains a potential nuclear localization signal between amino
acid residues 384 and 390 and is in fact localized to the cell nucleus
in transfected cells (Fig. 3). Third, like many transcription factors
with short half-lives (Chevaillier, 1993
), GKLF contains a PEST
sequence with a high PEST score of 5.8 (Rogers et al.,
1986
). Finally, GKLF contains abundant proline and serine residues,
amino acid residues purportedly involved in the transactivation
function of many transcription factors (Nakamura et al.,
1993
). Furthermore, its proline-rich nature is shared by proteins
closely related to GKLF, namely LKLF (Anderson et al.,
1995
), EKLF (Miller and Bieker, 1993
), and BTEB2 (Sogawa et
al., 1993
). Based on the high degree of homology in the
Krüppel region of the proteins and their proline-rich
characteristics, the latter three transcription factors have recently
been assigned to a new multigene family (Anderson et al.,
1995
). GKLF is thus the newest addition to this protein family.
Aside from the proline-rich domain of GKLF, amino acid sequence outside
its zinc finger region further defines its phylogenetic origin. For
example, absent from the GKLF sequence are the FAX
(
inger-
ssociated bo
) (Knochel
et al., 1989
) and KRAB
(
üppel-
ssociated
ox)
(Bellefroid et al., 1991
) motifs, the latter estimated to be
present in one-third of zinc finger-containing transcription factors.
Genes encoding the FAX- and KRAB-containing zinc finger proteins
(called class 1 zinc finger proteins by Pieler and Bellefroid (1994)
)
are generally organized in large clusters on certain chromosomes and
are thought to have appeared late during evolution. Most of these genes
are widely expressed in adult tissues and in most stages of
embryogenesis. The Krüppel family of transcription factors
(called class 2 zinc finger proteins by Pieler and Bellefroid (1994)
),
in contrast, includes fewer members that are highly conserved and that
function in the context of cell differentiation and embryogenesis. The
majority of class 2 zinc finger proteins exhibit highly restricted
patterns of expression in tissues and during embryogenesis. The
tissue-selective nature of GKLF and its close relationship
with other Krüppel-like factors such as LKLF and
EKLF indicate that it belongs to the class 2 zinc finger
protein gene family according to the classification of Pieler and
Bellefroid (1994)
.
Structural analysis of zinc finger-containing proteins indicates that
each finger consists of a
-pleated sheet at the amino-terminal half
and an
-helix at the carboxyl-terminal half and that the latter
makes direct contact with DNA (Berg, 1990
). A number of studies
examined the relationship between the amino acid sequence in the
helical portion of a finger and the DNA sequence to which the finger
binds (Berg, 1992
; Klug and Schwabe, 1995
; Nardelli et al.,
1991
). These studies revealed a number of important conclusions that
enable one to predict the DNA sequence to which a zinc finger protein
may bind. Table II compares the amino acid sequences in
the
-helical portions of each of the three zinc fingers of GKLF to
those of several other Krüppel-like transcription factors along
with the DNA sequences with which the fingers interact. It is apparent
that amino acid sequences in the helical region of each of the three
zinc fingers of GKLF, BTEB2, and EKLF are identical. Moreover, finger 2 of GKLF is identical to finger 1 of zif/268 and nearly identical to
finger 2 of Sp1 as well as finger 3 of zif/268. With the exception of
EKLF, these amino acid sequences recognize the consensus trinucleotide
GCG. In addition, finger 1 of GKLF is highly similar to finger 1 of
Sp1, which binds the trinucleotide GGG. Finally, finger 3 of GKLF is
very similar to finger 3 of Sp1 and finger 2 of zif/268, both of which
bind to the sequence GGG. Based on these comparisons, one can predict a
sequence of 5
-GGG GCG GGG-3
to which GKLF is potentially capable of
binding. This sequence is identical to the binding sequences of BTEB2
and Sp1. Of note is that although EKLF recognizes a different DNA
sequence despite an overall identity in the zinc finger sequences, it
is capable of interacting with an Sp1-binding site (Hartzog and Myers,
1993
).
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
The expression of GKLF in cultured fibroblasts is of
interest. The cDNA library from which GKLF was initially
derived was made with RNA from quiescent NIH 3T3 cells that had been
stimulated with serum for 3 h. Because the number of positive
clones obtained during a repeat screening of the same library with a
partial GKLF cDNA fragment was quite high (>50 positive
plaques out of a total of 1 million), we initially thought that
GKLF, like zif/268, would behave like an
immediately-early gene. We were therefore surprised to find from the
Northern blot experiment shown in Fig. 4 that GKLF behaved
quite differently from zif/268 or other immediate-early
genes. The differences include the following. 1) The steady-state
GKLF transcript level was high in serum-starved quiescent
NIH 3T3 cells. 2) The transcript level did not rise appreciably during
the first few hours of serum induction. 3) The transcript level began
to fall at 8 h of serum treatment. In addition, the
GKLF transcript was nearly undetectable in RNA harvested
from exponentially proliferating cells. Combining the results of serum
stimulation (Figs. 4 and 5), serum starvation (Fig. 6), and contact
inhibition (Fig. 7) experiments, it becomes apparent that expression of
GKLF is associated with cessation of cell growth. It is
especially of interest to note that in serum-stimulated cells, the
GKLF transcript level first decreases at a time that
immediately precedes the S phase of the cell cycle (Figs. 4 and 5)
(Quelle et al., 1993
). This observation suggests that GKLF
may exhibit a negative effect on cell cycle progression, particularly
at the G1/S transition phase. The diminished
[3H]thymidine incorporation by cells in which
GKLF was constitutively expressed (Table I) and the lack of
BrdUrd uptake by cells expressing GKLF (Fig. 8) support this
hypothesis.
The expression of GKLF in response to serum stimulation and
deprivation in cultured fibroblasts is reminiscent of that of a group
of genes exclusively expressed in the growth arrest state. These genes
are divided into two categories: the gas (
rowth
rrest-
pecific) genes (Ciccarelli et
al., 1990
; Gorski et al., 1993
) and the gadd
(
rowth
rrest- and
NA
amage-inducible) genes (Fornance et al., 1989
;
Zhan et al., 1994
). Like GKLF, expression of the
gas genes is highest in quiescent cells and is
down-regulated following mitogen stimulation. In particular, the time
course of expression of GKLF following mitogen addition to
quiescent cells (Figs. 4 and 5), serum deprivation (Fig. 6), and cell
contact (Fig. 7) is remarkably similar to that for gas1
(Ciccarelli et al., 1990
; Schneider et al., 1988
)
and gas5 (Ciccarelli et al., 1990). Similar to
the inhibitory effect of GKLF on DNA synthesis (Table I),
when ectopically expressed, some of the gas genes cause
growth arrest and are thought to be involved in a negative circuit that
governs growth suppression (Del Sal et al., 1992
).
Nevertheless, despite an overall similarity in the pattern of
expression of the gas gene family, they encode a diverse
group of protein products. For example, gas1 and
gas3 encode integral membrane proteins (Del Sal et
al., 1992
; Manfioletti et al., 1990
); gas2
encodes a protein of the microfilament network system (Brancolini
et al., 1992
); and gas6 encodes a secreted,
vitamin K-dependent protein that is a ligand for the Axl
receptor tyrosine kinase (Varnum et al., 1995
). The
gax gene (
rowth
rrest-specific
homeobo
) is a notable exception of the gas gene
family in that it encodes a homeobox-containing transcription factor
that is highly specific for vascular smooth muscle cells (Gorski
et al., 1993
). Finally, gadd153, the only
gadd gene with an identified function, is a member of the
C/EBP family of transcription factors and is the human homologue of the
murine CHOP-10 gene (Ron and Habener, 1992
).
In vivo, the expression of GKLF is highly
tissue-selective and is enriched in regions of the intestinal tract,
testis, and lung (Fig. 9). It is of interest to note that a partial
cDNA fragment encoding the putative rat homologue of
GKLF was identified in Sertoli cells that had been treated
with follicle-stimulating hormone (clone 59) (Hamil and Hall, 1994
).
Other than its induction by follicle-stimulating hormone, little
information regarding the expression of clone 59 is available. In
situ hybridization experiments should help clarify the cellular
origin of GKLF in the testis. It is clear from Figs. 10 and
11, however, that expression of GKLF in the colon is highly
enriched in the crypt epithelium. This finding is supported by the
cloning of a putative human homologue of GKLF from the
colonic mucosa (see ``Results''). Moreover, results of in
situ hybridization indicate that the GKLF transcript is
localized to a population of epithelial cells residing in the middle to
upper crypt region. This portion of the colonic crypt is thought to
consist of cells that have undergone growth arrest and that have begun
to differentiate into mature colonocytes as they emerge from the
proliferating compartment at the base of the crypt (Gordon et
al., 1992
). Thus, GKLF may play two potential physiological roles
in this environment. It can act either as a growth-suppressing gene
product that is involved in the growth arrest of epithelial cells as
they exit the base of the crypt or as a differentiation-promoting gene
product that is responsible for activating downstream genes that are
required for the differentiated epithelial phenotype. These two
functions are not mutually exclusive such that GKLF can potentially
serve both, in a manner similar to MyoD, which promotes both myogenic
differentiation and terminal withdrawal from the cell cycle (Halevy
et al., 1995
). Clearly, the exact function of GKLF in the
cell cycle and/or in terminal differentiation awaits further
examination.
We thank Dr. Lanahan for providing the NIH 3T3 cDNA library and the Genetics Institute for the PMT3 plasmid. We also thank Dr. Corey Mjaatvedt for assisting in the in situ hybridization.