©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification and Characterization of a Novel Cytokine-inducible Nuclear Protein from Human Endothelial Cells (*)

Wei Chu , Daniel K. Burns (§) , Robert A. Swerlick (1), David H. Presky (¶)

From the (1) Department of Inflammation/Autoimmune Diseases, Hoffmann-La Roche Inc., Roche Research Center, Nutley, New Jersey 07110 Department of Dermatology, Emory University School of Medicine, Atlanta, Georgia 30322

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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.


INTRODUCTION

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 (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.

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- 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.


EXPERIMENTAL PROCEDURES

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) .

Radioactively labeled cDNA prepared from unstimulated HUVEC poly(A)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 10cells 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.


RESULTS

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 10recombinant 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) .

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 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 10plaques 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).

To study the kinetics of induction of C-193 mRNA, HDMEC were stimulated with TNF- 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) .

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.


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.




DISCUSSION

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--stimulated HDMEC.

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- 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 IB (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.

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-, 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.

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, the precursor of NF-B p105 and p100, IB, and a family of proteins with IB-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) .

In addition to the ankyrin-like repeats, the IB-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 IB 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.

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 IB-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.

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.

  
Table: 20


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) X83703.

§
Present address: Dept. of Cell Physiology, Glaxo Research Institute, Research Triangle Park, NC 27709.

To whom correspondence should be addressed: Tel.: 201-235-3544; Fax: 201-235-5046.

The abbreviations used are: IL-1, 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.


ACKNOWLEDGEMENTS

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.


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