From the
Vascular endothelial cells undergo profound changes upon
cellular activation including expression of a spectrum of cell
activation-associated genes. These changes play important roles in many
physiological and pathological events. By differential screening of a
cDNA library prepared from interleukin-1
Endothelial cell activation has been implicated in many
physiological and pathological events including inflammation, immunity,
and atherosclerosis. In response to various inflammatory stimuli such
as interleukin-1
Identification of the genes
involved in the endothelial cell activation process is an important
first step in understanding the mechanism of activation at the
molecular level. Through differential screening of a cDNA library
prepared from IL-1
Radioactively labeled cDNA prepared from
unstimulated HUVEC poly(A)
One of the
positive clones isolated, designated as MEC-193, is approximately 0.5
kilobase long and appears to encode part of a 2.0-kilobase mRNA whose
expression is rapidly induced by IL-1
To study the
kinetics of induction of C-193 mRNA, HDMEC were stimulated with
TNF-
To verify the identified open
reading frame and to characterize the polypeptide product encoded by
C-193 mRNA, in vitro transcription and translation analysis
was performed. Plasmids C-193F and C-193-FLAG/pcDNAI were used as the
templates. As shown in Fig. 5 A, a predominant band was
detected from the in vitro translated product of each
construct at a position which is in agreement with the predicted
molecular weight for C-193 protein, demonstrating that the predicted
open reading frame of C-193 is able to direct the synthesis of C-193
protein. To examine whether the FLAG tag attached to the carboxyl
terminus of C-193 is functional, equivalent amounts of translation
products were immunoprecipitated with anti-FLAG monoclonal antibody M2
and analyzed by SDS-PAGE. As expected, the in vitro translated
C-193-FLAG protein was specifically immunoprecipitated from translation
products of construct C-193-FLAG/pcDNAI, but not C-193F, suggesting
that antibody M2 and C-193-FLAG protein could be used for further
characterization of C-193 protein.
Endothelial cells play important roles in many pathological
processes including inflammation, immunity, and atherosclerosis.
Knowledge of genes that are altered in expression upon endothelial cell
activation will help us understand the mechanisms of endothelial cell
involvement in these processes at the molecular level. In searching for
genes whose expression is induced during cytokine activation of HDMEC,
we have identified a novel gene, designated as C-193, by differential
screening of a cDNA library prepared from IL-1- and
TNF-
The rapid, protein synthesis-independent
induction of C-193 mRNA expression in endothelial cells after
cytokine-stimulation indicates that C-193 is a new addition to the
primary response gene family. Differential screening of cDNA libraries
has been used to identify a number of primary response genes induced in
response to cytokines or growth factors (for review, see Ref. 4). Many
genes of this family are transcription regulatory factors which are
involved in signal transduction and control of secondary gene
expression. The characteristic feature of this family is that gene
induction can occur in the absence of protein synthesis. Induction of
the C-193 gene by IL-1 or TNF-
Both C-193 mRNA and protein
contain instability elements (AU-rich motifs and PEST-rich sequence,
respectively) suggesting that C-193 may also be regulated at the level
of mRNA and protein stability, as seen in many short lived proteins
such as c- fos and c- myc. Homologous sequences to
C-193 gene were detected in many other species suggesting that C-193
gene is highly conserved during evolution. The expression of C-193 is
induced by IL-1, TNF-
An interesting feature of C-193 is the presence of four
tandem ankyrin-like repeats. The ankyrin-like repeat structure is a
33-amino-acid sequence motif with about one-third of the residues
highly conserved, which often appears in tandem arrays with variable
numbers of repeats. This type of structure has been observed in a group
of proteins with different functions and subcellular locations
(42) . A large number of proteins in this group are either
transcription factors or transcription factor inhibitors, including
GABP
In addition to the
ankyrin-like repeats, the I
The rapid,
protein synthesis-independent induction of C-193 after cytokine
stimulation, its location in the nucleus, the presence of ankyrin-like
repeat structure, and the similarities with I
In
summary, we have described the identification and characterization of a
novel cytokine-inducible nuclear protein, C-193. Gene expression of
C-193 is highly restricted to endothelial cells, and the induction of
C-193 gene expression by cytokines is protein synthesis-independent.
C-193 protein is capable of binding DNA cellulose and contains many
structural features commonly found in transcriptional regulators,
suggesting that C-193 may play a role in the regulation of gene
expression associated with endothelial cell activation. Further
characterization of the function of C-193 and its pattern of expression
will provide insights into its role in the mechanisms of endothelial
cell activation and gene regulation.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank/EMBL Data Bank with accession number(s) X83703.
We thank Dr. A. Goldstein, D. Gaizband, and G.
Schwinge for providing HUVEC, W. McComas and R. Motyka for
oligonucleotide synthesis, D. Larigan and J. Levine for DNA sequencing,
Dr. T. C. Tsang for the cytokine-induced HUVEC cDNA library, and J.
Best for assistance with HUVEC cDNA library screening.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and tumor necrosis
factor-
-stimulated human dermal microvascular endothelial cells,
we have identified a novel cytokine-inducible gene, designated as
C-193. The compiled cDNA sequence of C-193 is 1901 base pairs long and
shows no significant homology with any known gene sequence. Genomic DNA
analysis revealed that C-193 is encoded by a single gene, which is
conserved in different mammalian species. The C-193 gene was localized
to human chromosome 10 by Southern blot analysis of somatic cell
hybrids. Multiple AT-rich mRNA decay elements were identified in the
3`-untranslated region. C-193 mRNA expression was rapidly and
transiently induced by treatment with interleukin-1
or tumor
necrosis factor-
, reached a peak of expression about 16 h post
tumor necrosis factor-
stimulation, and the induction of C-193 was
protein synthesis independent. Lipopolysaccharide and cycloheximide
were also potent inducers of C-193 mRNA. Therefore, C-193 represents a
new addition to the primary response gene family. In vitro translation of C-193 yielded a 36-kDa protein product, consistent
with the predicted open reading frame of 318 amino acids and a
calculated molecular mass of 36 kDa for C-193 protein. The predicted
protein sequence contains a basic amino acid cluster similar to a
nuclear localization signal, four tandem repeats of ankyrin-like
sequence, and multiple consensus protein phosphorylation sites. C-193
was engineered with a FLAG tag at its carboxyl terminus and transiently
expressed in COS cells. Consistent with the presence of a putative
nuclear localization signal, the C-193-FLAG protein was localized to
the nucleus of transfected COS cells by indirect immunofluorescence
microscopy. C-193-FLAG prepared in vitro was capable of
binding DNA cellulose. These results indicate that C-193 protein may
play an important role in endothelial cell activation.
(IL-1
),
(
)
tumor
necrosis factor
(TNF-
), and bacterial lipopolysaccharide
(LPS), endothelial cells undergo profound changes in expression of a
spectrum of genes resulting in an activated phenotype
(1, 2, 3) . A number of genes have been
identified in cultured endothelial cells stimulated with TNF-
or
IL-1
and have been referred to as primary response genes. The
expression of these primary response genes is rapidly induced,
transient in nature, and transcriptional induction dose not require
de novo protein synthesis
(4) . These genes include a
number of cell surface adhesion molecules involved in recruiting
circulating leukocytes
(5) , chemotactic factors
(6, 7) , proteins involved in hemostasis
(8) ,
and a number of novel proteins with unknown functions
(9, 10) . Many of these primary response genes encode
transcription regulatory factors
(11) . These genes are of
interest because they are an important part of the response to
activation stimuli and function to propagate the activation signal by
controlling the expression of a set of secondary response genes in a
cell type or stimulus-specific fashion.
- and TNF-
-stimulated human dermal
microvascular endothelial cells (HDMEC), we have isolated a novel
serine-threonine protein kinase
(12) and numerous
cytokine-inducible sequences including cDNA clones encoding multiple
forms of human E-selectin
(13) . In this report, we describe the
isolation and characterization of a cDNA designated as C-193 which
encodes a novel, cytokine-inducible nuclear protein in endothelial
cells.
Endothelial Cell Cultures
HDMEC were isolated
from human neonatal foreskins and human umbilical vein endothelial
cells (HUVEC) were isolated from fresh umbilical cords by collagenase
digestion as described previously
(13, 14) . All cells
were used under five passages.
cDNA Library Construction and Screening
Total RNA
was extracted from IL-1- and TNF-
-stimulated HDMEC (500
units/ml each for 16 h) and unactivated HUVEC by the guanidine
isothiocyanate method
(15) . Poly(A)
RNA was
purified by two cycles of chromatography on oligo(dT)-cellulose
(16) . All cDNAs used for library construction and probe
synthesis were primed with oligo(dT) primer. A cDNA library was
prepared in pcDNAI (Invitrogen, San Diego, CA) from HDMEC stimulated
for 16 h with IL-1
and TNF-
by the method of Gubler and
Hoffman as described previously
(13, 17) . The library
was screened at 500 colonies/150-mm plate by differential hybridization
as described
(18) .
RNA was used as control
probe while cDNA prepared from cytokine-stimulated HDMEC
poly(A)
served as the induced probe. Colonies which
hybridized only to the induced probe were rescreened, and their DNA was
isolated and sequenced using an automated DNA thermocycle sequencer
(Applied Biosystems, Foster City, CA). To isolate the full-length cDNA
encoding C-193 sequence, the entire cDNA fragment of MEC-193 was used
as a probe to screen a cDNA library prepared in
SH lox (Novagen, Madison, WI) from HUVEC stimulated for 4 h with
IL-1
and TNF-
. Hybridization screening was carried out with
radioactively labeled MEC-193 insert in 6
SSC (1
SSC is
150 mM NaCl, 15 mM sodium citrate, pH 7.0), 5
Denhardt's solution, and 100 µg/ml salmon sperm DNA overnight
at 65 °C. After hybridization, the filters were washed in 0.1
SSC at 65 °C prior to autoradiography. The nucleotide
sequence was determined for both strands of positive clones isolated,
and the DNA sequence was compiled from the overlapping cDNA clones.
Sequence analysis was performed using the Genetics Computer Group
software package
(19) .
Northern Blot Analysis of C-193 Induction and
Expression
Northern blot analysis was performed according to
standard methods as described
(18) . RNA was isolated from
3
10
cells of each cell type using a micro RNA
isolation system from Pharmacia LKB Biotechnol. For studies examining
the kinetics of C-193 mRNA induction, subconfluent HDMEC or HUVEC were
grown in T-75 flasks, treated with the indicated inducer for the
specified time period, and then RNA was isolated as described above.
RNA samples were separated on a 1.2% agarose, formaldehyde gel and
transferred to a Nytran membrane (Schleicher & Schuell). The 5`
SphI restriction fragment of clone H193-2 was
radioactively labeled using a random hexanucleotide labeling kit
(Stratagene, La Jolla, CA) and used as a probe. The hybridization
conditions were 3
SSC, 50% formamide, 5
Denhardt's solution, 0.1% SDS at 42 °C. After hybridization
the filter was washed two times with 0.5
SSC, 0.1% SDS at 60
°C for 30 min each.
Plasmid Construction and in Vitro Transcription and
Translation
Plasmid construct C-193F, which contains near
full-length cDNA sequence of C-193, was prepared by exchange of the 5`
end SstI restriction fragment of H193-13 with the
corresponding fragment isolated from clone H193-21 using a
SstI site upstream to the cloning site in the pSH lox vector. C-193-FLAG/pcDNAI was constructed by inserting a
polymerase chain reaction fragment of the C-193 coding region only
(nucleotides 250-1206) into a pcDNAI vector. The polymerase chain
reaction was performed with plaque-forming units DNA polymerase
(Stratagene) and a set of primers as shown below:
5`-CGGGATCCGCCACCATGATGGTACTGAAAGTAGAGG-3` and
5`-GCTCTAGACTACTTGTCATCGTCGTCCTTGTAGTCGAATGTAGCTATGCGAGAGGT-C-3`. The
3` primer encodes an eight amino acid FLAG peptide tag
(20) to
facilitate subsequent detection and characterization of C-193-FLAG
recombinant protein. The orientation and DNA sequence were verified by
DNA sequencing of all constructs. The two DNA constructs C-193F and
C-193-FLAG were linearized with NotI 3` of the cloning site in
the vector and used as templates for in vitro transcription
with T7 RNA polymerase (Stratagene) and subsequent in vitro translation with rabbit reticulocyte lysate (Stratagene) in the
presence of [S]methionine (Amersham Corp.)
according to the manufacturers' instructions. Aliquots of the
in vitro translated products were used for
immunoprecipitation, DNA cellulose binding studies, and were analyzed
by SDS-PAGE. The gels were fixed with 40% methanol, 10% acetic acid for
30 min, treated with Enlightening (DuPont NEN) for 30 min, dried, and
exposed to x-ray film.
Expression and Detection of Recombinant C-193-FLAG
Protein in Transfected COS Cells
The polymerase chain reaction
fragment which contains the entire protein coding region of C-193 and a
FLAG tag attached to its carboxyl terminus as described above was blunt
ended and inserted into a mammalian expression vector pEF-BOS
(21) to yield the C-193-FLAG/pEF-BOS expression construct. The
proper orientation of the insert and its DNA sequence were confirmed by
DNA sequencing. This DNA was introduced into COS cells by DEAE-dextran
transfection as described previously
(12) . The transfected
cells were cultured at 37 °C in Iscove's modified
Dulbecco's medium containing 10% fetal calf serum for 48-72
h before harvesting. For immunofluorescence staining, COS cells
transfected with either C-193-FLAG/pEF-BOS or control pEF-BOS DNA were
fixed in 2% paraformaldehyde for 10 min and incubated sequentially with
a PBS solution containing 10 mM glycine for 10 min and PBS
solution with 1% Triton X-100. Immunofluorescence staining was
performed using monoclonal anti-FLAG antibody M2 (Eastman Kodak Co.) at
5 µg/ml and the F(ab`)fragment of affinity purified,
rhodamine-conjugated goat anti-mouse IgG (Cappel, West Chester, PA).
The stained cells were washed and viewed under a Zeiss epifluorescence
microscope. For immunoprecipitation and Western blot analysis of the
C-193-FLAG protein, COS cells were transfected with the expression
construct C-193-FLAG/pEF-BOS or control pEF-BOS vector DNA alone by
electroporation using a Bio-Rad Gene Pulser System as described
(22) . The transfected cells were scraped from the plates after
72 h and washed with PBS. Cytoplasmic and nuclear extracts for each
transfectant were prepared by the methods of Dignam et al. (23) . Cell extracts, normalized to the same cell
equivalents, were diluted with CHAPS lysis buffer (50 mM
Tris-HCl, pH 7.4, 10 mM CHAPS, 100 mM NaCl, 10
mM iodoacetamide, and 1 mM phenylmethylsulfonyl
fluoride) and incubated with 5 µg of M2 antibody and 50 µl of
GammaBind G Sepharose (Pharmacia) for 4 h at 4 °C. The
immunoprecipitates were washed four times with CHAPS lysis buffer,
boiled in SDS sample buffer, and aliquots were separated by SDS-PAGE.
The gel was electrotransfered onto a Nitrocellulose membrane for
Western blot analysis. The membrane was blocked in Tris-buffered saline
solution containing 5% non-fat dry milk, incubated with M2 antibody at
5 µg/ml followed by an affinity purified, peroxidase-conjugated
goat anti-mouse IgG (Boehringer Mannheim), and developed using the ECL
system (Amersham) following the manufacturer's instructions.
FLAG-tagged bacterial alkaline phosphatase (BAP-FLAG) was included as a
control for the Western blot process. Equivalent amounts of the same
extracts were also subjected to Western blot analysis with a rabbit
anti-GAPDH antisera (Chemicon International Inc., Temecula, CA),
followed by peroxidase-conjugated goat anti-rabbit IgG as described
above.
Southern Hybridization Analysis
Human genomic DNA
(10 µg each, Clontech Laboratories Inc.) was digested to completion
with the indicated restriction enzymes (Boehringer Mannheim). The
cleaved DNA was separated by electrophoresis on a 0.8% agarose gel and
transferred onto a Nytran membrane. The filter was probed with the 5`
end of SphI restriction fragment of clone H193-2 as
described previously for Northern hybridization.
Determination of C-193 Gene Chromosomal Location
A
Southern blot containing genomic DNA from a panel of human/hamster
hybrid cell lines (Bios Corporation, New Haven, CN) was probed with the
5` SphI restriction fragment of clone H193-2 to
determine the chromosomal location of the C-193 gene. The chromosomal
assignment obtained by the above hybrid blot was further confirmed by
using polymerase chain reaction to amplify a 300-base pair long C-193
gene-specific fragment from DNA of human/rodent somatic cell hybrids
obtained from Coriell Cell Repositories (Camden, NJ).
DNA Cellulose Chromatography
Equivalent amounts of
[S]methionine-labeled C-193-FLAG protein
prepared by in vitro translation as described above was
applied to either a 2.0-ml DNA cellulose column (containing
0.25-1.5 mg of native calf thymus DNA/ml of Whatman CF cellulose,
Pharmacia) or a Whatman CF-1 cellulose column in column buffer (20
mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM
dithiothreitol, 50 µg/ml bovine serum albumin, 10% glycerol, and 1
mM phenylmethylsulfonyl fluoride) containing 50 mM
NaCl. The flow-through was reapplied to the columns, which were then
washed twice each with one column volume of column buffer containing 20
mM NaCl. The columns were step eluted sequentially with two
column volumes of column buffer containing 0.1, 0.2, 0.5, and 1.0
M NaCl. A portion of each fraction (100 µl) was mixed with
3 ml of Aquasol scintillation fluid (Eastman Kodak Co.) and counted in
a Beckman LS7800 liquid scintillation counter. For immunoprecipitation
analysis, each fraction of the DNA cellulose column was adjusted to
CHAPS lysis buffer conditions (described above) and immunoprecipitated
with anti-FLAG antibody M2 followed by SDS-PAGE and autoradiography as
described above.
Cloning and Sequencing of C-193
In order to
identify genes preferentially expressed in cytokine-activated HDMEC, a
cDNA library was constructed using mRNA isolated from HDMEC stimulated
for 16 h with IL-1 and TNF-
and screened by differential
hybridization as described under ``Experimental Procedures.''
Duplicate filter lifts of approximately 4
10
recombinant colonies were hybridized with radioactively labeled
cDNA probes generated from mRNA isolated from either 16-h IL-1
-
and TNF-
-stimulated HDMEC (induced probe) or unstimulated HUVEC
(control probe). Because the availability of the primary HDMEC is very
limited, we took advantage of the similarities between HUVEC and HDMEC
and used HUVEC cDNA as the control probe. Thus, positive clones which
hybridized only to the induced probe represent either true
cytokine-inducible genes or genes not induced by cytokine but expressed
at higher levels in HDMEC. These positive clones were rescreened and
their DNA isolated for further characterization. Among those positive
clones isolated were multiple forms of human E-selectin cDNA, an
endothelial cell-specific cell surface glycoprotein expressed only in
activated endothelial cells
(6, 13) .
or TNF-
in both HDMEC
and HUVEC as indicated by Northern blot analysis (see below). To
isolate the full-length sequence, the insert of MEC-193 was used as a
probe for screening a second cDNA library prepared from HUVEC
stimulated with IL-1
and TNF-
for 4 h. After screening a
total of 3
10
plaques at high stringency, about 90
hybridization-positive plaques were identified, which represents about
0.03% of the total mRNA population of cytokine activated HUVEC. Four
overlapping clones were subsequently isolated, and their DNA was
sequenced. Sequence analysis confirmed that these clones overlap the
original MEC-193 sequence (Fig. 1). The compiled sequence
consists of 1901 base pairs which end with a short poly(A) stretch. The
lack of a typical polyadenylation signal preceding the poly(A) sequence
suggests that the poly(A) stretch may be an internal sequence.
Interestingly, multiple mRNA decay elements, ATTTA, are found in the
3`-untranslated region. These ``AU-rich'' elements are
commonly found in mRNAs of cytokines, proto-oncogenes, and primary
response genes and are implicated in the rapid turnover of the
corresponding mRNA
(24) . A single open reading frame, beginning
with the ATG at nucleotide 250, was identified which encodes a protein
with 319 amino acid residues, designated as C-193. There is an in-frame
stop codon, TGA, found at bases 55-57 upstream of the initiation
sequence of the open reading frame (Fig. 1), further supporting
the idea that the identified putative open reading frame encodes a
full-length protein. A search of current DNA and protein data bases
(GenBank, release 84.0, and Swiss-PROT, release 29.0) with C-193
nucleotide or amino acid sequences failed to identify any known
sequences with significant homology.
Figure 1:
The cDNA structures and the
consensus nucleotide and the deduced amino acid sequences of C-193.
Upper panel, schematic diagram of composite C-193 cDNA
structure and the five overlapping cDNA clones. The rectangle shows the open reading frame, and the dotted area represents the nucleotides coding for the four ankyrin-like
repeats. Solid lines attached to the rectangle indicate 5`- and 3`-untranslated regions. The putative nuclear
localization signal ( NLS) is indicated by a vertical
bar. Four AT-rich mRNA destabilizing elements are shown by
arrows. The consensus phosphorylation sites are indicated by
Ps. Center panel, nucleotide sequence and predicted
protein structure of C-193. The nucleotides are numbered to the left of
the figure. Amino acid numbering begins with the first in-frame ATG at
base 250 and is located to the right of the figure. An in-frame stop
codon, TGA, located at nucleotide 195-197 upstream of the
putative initiation sequence is double-underlined. Sequences
containing mRNA destabilizing elements, ATTTA, in the 3`-untranslated
region are underlined. The putative nuclear localization
signal sequence KKRKK is shown in bold. The arrows indicate the domain containing four tandem ankyrin-like repeats.
The dashed lines underlining the sequence contains consensus
protein phosphorylation sites. Lower panel, sequence alignment
of the ankyrin-like repeats of C-193 and comparison of the consensus
sequence with that of NF-B p105 (43) and ankyrin (25). Numbers
in parenthesis after each repeat indicate the position of amino
acid residues, and numbers in parenthesis after NF-
B and
ankyrin indicate the number of ankyrin-like
repeats.
Gene Expression and Induction of C-193
Northern
blot analysis with RNA isolated from various human cell lines indicated
that C-193 expression is mainly restricted to activated vascular
endothelial cells, both microvascular and large vessel, as represented
by HDMEC and HUVEC, respectively (Figs. 2 and 3). C-193 gene expression
is rapidly and dramatically induced in both HDMEC and HUVEC after
stimulation with IL-1, TNF-
, and LPS. TNF-
seems to be
the most potent inducer of C-193 in both types of cells. A significant
amount of C-193 mRNA expression has been consistently observed in
unstimulated HUVEC (Fig. 3). However, this basal expression can
be further increased upon stimulation. In contrast, very low levels of
expression were seen in unstimulated HDMEC detectable only after a
prolonged exposure (data not shown).
Figure 3:
Northern blot hybridization analysis of
C-193 induction and expression in HUVEC and cultured human cell lines.
A, an autoradiogram of a Northern blot containing RNAs from
various human cell lines probed with radioactively labeled C-193 cDNA.
Total RNA (20 µg each) isolated from HUVEC ( lane 1), HUVEC
stimulated with IL-1 and TNF-
for 4 h ( lane 2),
HDMEC ( lane 3), HDMEC stimulated with IL-1
and TNF-
for 16 h ( lane 4), HeLa ( lane 5), 5637 ( lane
6), Hep G2 ( lane 7), MRC-5 ( lane 8), and C-32
( lane 9) were separated by electrophoresis on a 1.2%
agarose/formaldehyde gel and transferred onto a Nytran membrane. The
filter shown was exposed for 16 h at
80 °C with one
intensifying screen. The migration positions of the 28 S and 18 S
ribosomal RNAs are indicated. Lower panel, the same filter was
subsequently hybridized with a radioactively labeled GAPDH cDNA as a
measure of RNA loading. B, cytoplasmic RNA was isolated from
HUVEC treated with different stimuli as indicated for 4 h and subjected
to Northern blot analysis as described under ``Experimental
Procedures.'' The filter was sequentially probed with
radioactively labeled C-193 cDNA fragment ( upper panel) and a
GAPDH cDNA ( lower panel).
The induction by cytokine
stimulation observed in endothelial cells was not affected by the
addition of the protein synthesis inhibitor CHX, suggesting ongoing
protein synthesis is not required for induction. Furthermore, CHX alone
is able to cause significant induction of C-193 mRNA expression in both
HDMEC and HUVEC. Except for a low level of expression detected in
unstimulated Hep G2 cells, no C-193 expression was detected in any
other types of unstimulated cells examined including epithelial cells
(HeLa), bladder carcinoma cells (5637), fibroblasts (MRC-5), and
melanoma cells (C-32) (Fig. 3). C-193 expression was not detected
in either IL-1, TNF-, or calcium ionophore A23187 stimulated or
resting cells of hematopoietic lineage including the YT and Kit225 cell
lines (data not shown). C-193 expression was detected in
TNF-
-stimulated MRC-5 cells (data not shown).
for various times, and their cytoplasmic RNA was isolated for
Northern blot analysis with a C-193 cDNA fragment as probe. As shown in
Fig. 4
, no detectable C-193 mRNA is found in the unstimulated
HDMEC. However, upon treatment with TNF-
, C-193 expression is
rapidly induced within 30 min and reaches a maximum level after about
16 h.
Figure 4:
Time course of C-193 mRNA induction in
HDMEC in response to TNF- stimulation. A,
poly(A)
RNA samples were prepared from HDMEC harvested
after treatment with TNF-
for the times indicated and analyzed by
Northern blot hybridization. The blot was sequentially probed with
radioactively labeled C-193 cDNA ( upper panel) and GAPDH
( lower panel). The migration positions of bands containing the
28 S and 18 S ribosomal RNAs are indicated. Both autoradiograms shown
were exposed for 7 h at
80 °C with one intensifying screen.
B, the strength of hybridization signals for each time point
was quantitated by a PhosphorImager, normalized with respect to the
corresponding signals for GAPDH, and plotted as radioactivity unit
versus time (hours).
Characterization of the C-193 Protein
The deduced
C-193 protein contains 319 amino acid residues with a calculated
molecular mass of 36 kDa and a pI of 7.10. There is no apparent signal
peptide or recognizable hydrophobic transmembrane region in this
protein. Sequence analysis of the predicted amino acid sequence using
the Wordsearch program indicated a domain containing four tandem
ankyrin-like repeats
(25) located close to the carboxyl
terminus of the protein (Fig. 1). In addition, a cluster of basic
residues, KKRKK, was found preceding the ankyrin-like repeats. This
type of basic residue cluster has been implicated in intracellular
protein trafficking into the nucleus and is referred to as a nuclear
localization signal
(26, 27, 28) . Other
interesting features include a putative Tyr phosphorylation site at
amino acid 33 and a sequence containing multiple putative Ser and Thr
phosphorylation sites present at the extreme carboxyl terminus,
suggesting that protein phosphorylation may play a role in regulation
of C-193 protein function. A PEST-rich (52%) region at residues
108-126 was also identified. The PEST-rich sequence is thought to
confer susceptibility to rapid intracellular degradation and is
expressed in many short-lived proteins, including c- myc,
c- fos, and p53
(29) .
Figure 5:In vitro transcription and
translation of C-193. A, an autoradiogram showing the in
vitro translated products of C-193. Constructs C-193F and
C-193-FLAG/pcDNAI, which contain near full-length C-193 cDNA sequence
and FLAG-tagged C-193 coding region, respectively, were used as the
templates for in vitro transcription with T7 RNA polymerase
and subsequent in vitro translation with rabbit reticulocyte
lysate in the presence of [S]methionine. The
in vitro translation products of C-193F ( lane 1) and
C-193-FLAG/pcDNAI ( lane 2) were analyzed by SDS-PAGE on a 10%
gel followed by autoradiography. Positions of prestained molecular mass
standards are indicated on the left. B, immunoprecipitation of
in vitro translated C-193 products with anti-FLAG antibody.
The in vitro translated products were subjected to
immunoprecipitation with anti-FLAG antibody M2. The immunoprecipitates
were analyzed by SDS-PAGE on a 4-20% gel followed by
autoradiography. Notations are the same as in panel A. Both
autoradiograms were exposed for 16 h.
Expression of C-193 in COS Cells and Subcellular
Localization
A polymerase chain reaction fragment containing
C-193 coding region and a FLAG tag at the carboxyl terminus was
inserted into an expression vector pEF-BOS generating
C-193-FLAG/pEF-BOS. To determine the subcellular distribution pattern
of the C-193-FLAG protein, indirect immunofluorescence staining and
immunoprecipitation experiments with anti-FLAG antibody were performed
using COS cells transfected either with C-193-FLAG/pEF-BOS or pEF-BOS
vector DNA alone. As shown in Fig. 6 A, C-193 protein is
abundantly expressed and sequestered predominantly within the nucleus
of COS cells transfected with C-193-FLAG/pEF-BOS ( left panel).
No fluorescent staining was detected in COS cells transfected with the
vector DNA as control ( right panel). To further confirm its
nuclear localization, immunoprecipitation of C-193-FLAG protein
directly from nuclei isolated from transfected COS cells was carried
out. As shown in Fig. 6 B, cytoplasmic ( lane 1)
and nuclear ( lane 2) extracts were prepared from COS cells
transfected with C-193-FLAG/pEF-BOS or pEF-BOS vector DNA alone
(control DNA). The immunoprecipitates were separated by electrophoresis
on a 4-20% SDS-PAGE, and then transferred onto a nitrocellulose
membrane. The membrane was incubated with anti-FLAG antibody M2
followed by a horseradish peroxidase-conjugated secondary antibody and
visualized with an ECL system. Consistent with the immunofluorescence
results, the intact C-193-FLAG protein was detected in the nuclear
fraction of C-193-FLAG/pEF-BOS transfected COS cells, although there
was still a significant amount of protein that remained in the
cytoplasmic fraction, probably due to ongoing protein synthesis.
Interestingly, there were two additional bands present in the
cytoplasmic fraction that did not appear in the nuclear fraction. These
two bands appear to be degradation products of C-193-FLAG protein,
which may have lost the amino-terminal sequence containing the nuclear
localization signal. No band was evident in either fraction of COS
cells transfected with control pEF-BOS DNA. To ensure that the
C-193-FLAG protein detected in the nuclear fraction was not due to
contamination with cytoplasmic contents during preparation of the
nuclei, a Western blot for glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), a cytoplasmic enzyme, was performed using the same cell
extracts used for the C-193-FLAG immunoprecipitation
(Fig. 6 C). GAPDH was detected in cytoplasmic fractions
from both control DNA and C-193-FLAG/pEF-BOS transfected COS cells. No
detectable levels of GAPDH were seen in either nuclear fraction. These
results demonstrate that, consistent with the presence of a nuclear
localization signal, C-193-FLAG is indeed a nuclear protein.
Figure 6:
Subcellular localization of C-193-FLAG
protein in transfected COS cells. A, immunofluorescence
staining of transfected COS cells. COS cells were transfected with
either C-193-FLAG/pEF-BOS construct DNA ( left panel) or
pEF-BOS vector DNA alone ( right panel) as described under
``Experimental Procedures.'' The cells were subjected to
indirect immunofluorescence staining with anti-FLAG antibody M2
followed by a rhodamine-conjugated goat anti-mouse IgG 72 h after
transfection. B, immunoprecipitation of C-193-FLAG from
nuclear extracts of transfected COS cells. COS cells were transfected
with either plasmid pEF-BOS DNA alone (control DNA) or plasmid
C-193-FLAG/pEF-BOS (193/pef-bos) and incubated for 72 h. The
cytoplasmic extracts ( lane 1) and nuclear extracts ( lane
2) were prepared, immunoprecipitated with M2 antibody, separated
by SDS-PAGE on a 4-14% gel, and subjected to Western blot
analysis with M2 antibody as described under ``Experimental
Procedures.'' A FLAG-tagged bacterial alkaline phosphatase
( BAP-Flag) was used as a control for the Western blot process.
The arrow indicates the full-length C-193-FLAG protein. The
top and bottom bands are the IgG heavy chain and
light chain, respectively. Molecular mass markers, in kDa, are
indicated on the right. C, analysis of GAPDH
partitioning. Equivalent amounts of the same extracts used for
immunoprecipitation analysis of C-193 as described in B were
subjected to Western blot analysis for GAPDH as described under
``Experimental Procedures.'' The notations are the same as
described in B.
Genomic DNA Analysis and Chromosomal Assignment of C-193
Gene
Southern blot analysis of human genomic DNA digested with
various restriction enzymes suggested that C-193 is encoded on a single
locus in the human genome (Fig. 7 A). To determine the
extent of conservation of the C-193 gene, a Southern blot containing
PstI-digested genomic DNA from various species was hybridized
with a C-193 cDNA fragment at high stringency. The results are shown in
Fig. 7B, where a single hybridizing band is detected in
mouse, hamster, monkey, and rabbit, but no hybridization was observed
in yeast and Drosophila, indicating that the C-193 gene is
highly conserved across different mammalian species. Using a panel of
somatic cell hybrids, the C-193 gene was unambiguously localized to
human chromosome 10 (). This was further confirmed by
polymerase chain reaction analysis of somatic cell hybrid DNA obtained
from an independent source (data not shown).
Figure 7:
Southern blot analysis of human genomic
DNA and conservation of the C-193 gene in different species.
A, human placenta DNA (10 µg each) was digested with
restriction enzymes EcoR I ( E), HindIII
( H), or PstI ( P), separated by
electrophoresis on a 0.8% agarose gel, and blotted onto a Nytran
membrane. The filter was probed with radioactively labeled 5` end
SphI restriction fragment of clone H193-2 in 3
SSC, 50% formamide at 42 °C, and washed in 2
SSC, 0.1% SDS
at 60 °C two times for 30 min each. The autoradiogram shown was
exposed for 48 h at
80 °C with one intensifying screen.
B, genomic DNA (10 µg each) from the indicated species was
digested with PstI and analyzed by Southern blotting under the
same conditions as described for panel
A.
In Vitro DNA Binding Activity of C-193 Protein
To
test the DNA binding activity of C-193, in vitro translated
S-labeled C-193-FLAG protein was applied to a native calf
thymus DNA cellulose column or, as a control, a cellulose column of the
same type as the DNA cellulose column. These two columns were then
washed and eluted with increasing concentrations of salt. As shown in
Fig. 8
, virtually all of the C-193-FLAG protein applied to the
cellulose column was collected in the flow-through and the first two
wash fractions. In contrast, a significant amount of the C-193-FLAG
protein bound to the DNA cellulose column. This bound protein eluted at
low concentrations of NaCl (0.1-0.5 M NaCl). To confirm
that these eluted fractions from the DNA cellulose column contained
C-193-FLAG protein, the column fractions were immunoprecipitated with
anti-FLAG antibody and the immunoprecipitates were analyzed by
SDS-PAGE.
S-Labeled C-193-FLAG contained in the gel was
quantified with a PhosphorImager. As shown in Fig. 8, the
C-193-FLAG protein was detected in fractions from the second 0.1
M NaCl elution to the 0.5 M NaCl eluted fractions, in
agreement with the analysis by scintillation counting. These results
demonstrate that C-193-FLAG does not bind to cellulose but is capable
of binding to DNA cellulose.
Figure 8:
DNA
cellulose chromatography of in vitro prepared
[
S]C-193-FLAG protein. A,
chromatography of C-193-FLAG protein on cellulose and DNA cellulose
columns. Equivalent amounts of in vitro prepared
[
S]C-193-FLAG protein were applied to each
column. About 5% of each column fraction (100 µl) of flow-through
( FT1 and 2), washes ( W1, 2, and
3), and elutions with 0.1, 0.2, 0.5, and 1.0 M NaCl
in column buffer was analyzed by scintillation counting. B,
immunoprecipitation analysis of C-193-FLAG protein in DNA cellulose
column fractions with anti-FLAG antibody M2. Each fraction was adjusted
to CHAPS lysis buffer conditions and incubated with 5 µg of M2
antibody and 50 µl of GammaBind G-Sepharose. The immunoprecipitates
were analyzed by SDS-PAGE on a 12% gel followed by autoradiography. An
equivalent amount of [
S]C-193-FLAG protein as
applied to the column was included as a control ( lane L).
Molecular mass standards are indicated on the
left.
-stimulated HDMEC.
was not affected by the addition of
the protein synthesis inhibitor CHX. In fact, CHX alone is a potent
inducer of C-193 mRNA expression. It has been shown that CHX is able to
increase steady-state levels of many other primary response gene or
cytokine RNAs, probably by a combination of stimulating transcription
and stabilizing the mRNA
(30, 31, 32, 33, 34, 35, 36) .
For example, CHX has been shown to induce E-selectin expression in
HUVEC, in part by increasing NF-
B-dependent transcription
(30) . It has been suggested that activation of NF-
B
activity by CHX is probably due to the inhibition of the synthesis of a
rapidly turning-over negative factor such as I
B
(37, 38, 39) . Moreover, the NF-
B binding
sequence element in the ELAM-1 promotor was found to be necessary and
sufficient to confer the CHX-dependent inducibility
(30) . C-193
expression is induced by IL-1, TNF-
, and LPS, all of which
activate transcription factor NF-
B, suggesting that C-193 gene
expression, like many other primary response genes, may also be
regulated by the NF-
B pathway.
, or LPS in both small vessel and large
vessel endothelial cells, as represented by HDMEC and HUVEC,
respectively. Unstimulated HUVEC exhibited a significant level of basal
expression of C-193 mRNA which was further increased upon cytokine
stimulation. In contrast, basal expression of C-193 mRNA was detectable
in unstimulated HDMEC only after prolonged exposure of the
autoradiograph. This difference in the basal expression levels of C-193
between HDMEC and HUVEC may reflect the heterogeneity among different
types of endothelial cells. Recent studies have demonstrated that
heterogeneity between HDMEC and HUVEC exists in many respects such as
growth requirements in vitro, prostaglandin production, cell
surface markers, and the regulation of cell adhesion molecules
(40, 41) . In IL-1- and TNF-
-activated HUVEC, the
C-193 transcripts were detected at high levels as determined by
Northern blot hybridization (Fig. 3 A) and represent
0.03% of the total mRNA population, as revealed during the secondary
cDNA library screening. Such high level expression indicates that C-193
may play a critical role in endothelial cell activation. C-193
expression was not found in many other types of cells examined,
suggesting that C-193 may function in a endothelial cell type-specific
manner.
, the precursor of NF-
B p105 and p100, I
B, and a
family of proteins with I
B-like activities isolated from diverse
species such as MAD-3, pp40, ECI-6, RL/IF-1, and bcl-3
(42, 43, 44, 45, 46, 47, 48, 49) .
Although the precise role for the ankyrin-like repeats in these
proteins remains largely unknown, it is speculated that they may be
involved in interactions between distinct proteins. It has been shown
that the four ankyrin-like repeats in the amino terminus of the
transcription factor subunit GABP
are required for its interaction
with the GABP
subunit to form a stable and functional high
affinity DNA binding complex
(43) . Furthermore, these repeats
can be cross-linked to DNA when GABP is in association with its target
DNA sequence. In addition, it has been shown that ankyrin-like repeats
are also capable of mediating interactions between regions of a single
protein such as in NF-
B p105
(50) .
B-like proteins share many features
including similar size, five to seven ankyrin-like repeats which
account for about 50% of the total protein mass, and multiple consensus
protein phosphorylation sites. Expression of most of these proteins
(MAD-3, ECI-6, and RL/IF-1) has been shown to be up-regulated upon cell
activation
(45, 47, 48) . For example, ECI-6,
also a primary response gene, is inducible in endothelial cells by
IL-1, TNF-
, and LPS through an NF-
B-dependent pathway
(47) . C-193 contains many similarities to this I
B protein
family including protein size, induction profile, ankyrin-like repeats
which account for about 41% of the total protein mass, and multiple
protein phosphorylation sites suggesting that C-193 may exhibit similar
biological functions and may play a role in regulation of gene
expression associated with endothelial cell activation.
B-like proteins
suggest that C-193 protein may play a role in the regulation of gene
expression. This hypothesis is supported by the evidence presented here
for a DNA binding activity of C-193 protein. C-193-FLAG protein
specifically bound to a DNA cellulose column. The bound C-193-FLAG
protein was eluted off the DNA cellulose at a relatively low salt
concentration, suggesting the DNA binding affinity of C-193-FLAG
protein is low. Although we have ruled out C-193-FLAG protein binding
to cellulose, we cannot completely rule out the possibility of
nonspecific binding to the DNA cellulose. However, the observed low
affinity of C-193 protein binding to DNA cellulose may reflect the
following: (i) the presence of four ankyrin-like repeats raises the
possibility that the function of C-193 protein may be regulated by
direct interactions with other protein(s) through the ankyrin-like
repeat structure, as has been shown for GABP
(43) . Higher
affinity DNA binding may require interaction with additional
subunit(s). (ii) Post-translational processing, e.g. protein
phosphorylation, is known to have regulatory effects, both positive and
negative, on the DNA binding activity of many transcription factors and
DNA-binding proteins
(51, 52, 53, 54, 55) . For
example, inhibition of DNA binding activity by protein phosphorylation
has been observed for a number of transcription factors including IP-1,
CREB, and c-Jun
(51, 52, 56) . In other cases,
protein phosphorylation can increase the DNA binding affinities of
transcription factors including SRF and Egr-1
(54, 55) .
C-193 contains multiple consensus sites for both tyrosine and
serine-threonine phosphorylation. Therefore, it is possible that the
degree of phosphorylation of C-193 protein at these consensus protein
phosphorylation sites may also contribute to the observed low affinity
binding to DNA cellulose. (iii) Sequence-specific DNA binding of
transcription factors is clearly estatablished
(57) . C-193
protein may bind with high affinity to a very restricted sequence of
DNA, which may not be present on the DNA cellulose column. It is also
possible that the addition of the FLAG tag at the carboxyl-terminal of
C-193 may reduce the DNA binding activity of C-193 protein.
, interleukin-1
; HUVEC, human
umbilical vein endothelial cells; HDMEC, human dermal microvascular
endothelial cells; TNF-
, tumor necrosis fator-
; CHX,
cycloheximide; PBS, phosphate-buffered saline; GAPDH,
glyceraldehyde-3-phosphate-dehydrogenase; LPS, lipopolysaccharide;
PAGE, polyacrylamide gel electrophoresis; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic
acid.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.