THE OF ,267, NO. 2, Issue of pp. 1317-1326.1992 1992 by ...shock, cachexia, and cerebral malaria (for review see Ref. 2). Recently, the proinflammatory effects of TNF mediated pri-

THE JOURNAL OF BIOLOGICAL CHEMISTRY 8 1992 by The American Society for Bioehemistry and Molecular Biology, Inc.

,267, NO. 2, Issue of January 15, pp. 1317-1326.1992 Printed in U. S. A .

Characterization of a Novel Tumor Necrosis Factor-a-induced Endothelial Primary Response Gene*

(Received for publication, June 27, 1991)

Frederick W. Wolfs, Rory M. Marks#, Vidya SarmaS, Mary G . Byersll, Ronald W. KatzS, Thomas B. Showsll, and Vishva M. DixitSII From the Departments of $Pathology and §Internal Medicine, The University Of Michigan Medical Center, Ann Arbor, Michigan 48109 and the TDepartment of Human Genetics, Roswell Park Memorial Institute, Buffalo, New York 14263

The response of endothelial cells to the cytokine tumor necrosis factor-a (TNF) is complex, involving the induction and suppression of multiple genes and gene products. Differential screening of a TNF-stimulated, cycloheximide-treated human umbilical vein endothelial cell library has resulted in the cloning of several novel cDNAs whose protein products are involved in the primary response of the endothelium to TNF. One of these cDNAs, designated B12, is further characterized here. B12 is encoded by a 3.5-kilobase transcript and is induced rapidly and transiently by TNF. Tran- script expression is found to be developmentally regulated in a tissue-specific manner, with B12 message being differentially expressed in the heart and liver during mouse embryogenesis. The open reading frame of B12 predicts a 316-amino acid sequence rich in charged residues, particularly at the carboxyl terminus, and has neither significant homology to other known proteins nor to any extant sequence motifs. B12 is found to be a highly conserved single-copy gene which is located in the q22dq23 region of human chromosome 17. Polyclonal antibodies raised against a large portion of the B12 open reading frame immunoprecipitate a 36-kilodalton polypeptide from wheat germ lysates programmed to translate in vitro transcribed B12 mRNA. The B12 protein is further shown to be induced in human umbilical vein endothelial cells by TNF, and the protein is shown to be rapidly degraded.

Tumor necrosis factor (TNF)-a’ is a proinflammatory cytokine secreted predominantly by activated macrophages and was first described as an agent leading to the necrosis of certain tumors (1). Since then a wide range of pathologic conditions have been linked to TNF, including endotoxic

* This work was supported by Grant HL45351 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be 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 GenBankTM/EMBL Data Bank with accession number(s) M80783.

11 Established Investigator of the American Heart Association. To whom correspondence should be addressed Dept. of Pathology, The University of Michigan Medical School, 1301 Catherine St., Box 0602, Ann Arbor, MI 48109-0602.

’ The abbreviations used are: TNF, tumor necrosis factor; IL, interleukin; HUVE, human umbilical vein endothelial; CHX, cycloheximide; LPS, lipopolysaccharide; SDS, sodium dodecyl sulfate; TES, 2-([2-hydroxy-l,l-bis(hydroxymethyl)ethyl]amino~ethane- sulfonic acid; PCR, polymerase chain reaction; bp, base pair(s).

shock, cachexia, and cerebral malaria (for review see Ref. 2). Recently, the proinflammatory effects of TNF mediated pri- marily by its ability to activate leukocytes and vascular cells has been the focus of much research (3,4).

A primary target for TNF is the vascular endothelium, which is located at the interface of the bloodstream and body tissues. TNF-stimulated endothelium actively participates in mediating a proinflammatory response as it promotes leukocyte chemotaxis and adherence to the endothelial cell surface (5), induces an alteration of the normally anticoagulant cell surface to one that supports coagulation (6-9), and causes secretion of paracrine factors such as interleukin-lp (IL-lp), interleukin-6 (IL-6), and interleukin-8 (IL-8) that amplify the inflammatory response (10-12). At high doses TNF is growth inhibitory to endothelial cells (13) and is capable of inducing apoptosis or programed cell death (14), and this may further contribute to tissue injury.

The molecular mechanisms responsible for the effects of TNF on the endothelium are complex and as yet are only partially understood. This is especially true of those genes that constitute the initial genetic response to TNF. Such genes, known as primary response genes, are induced in the absence of intervening protein synthesis and generally encode proteins which initiate alterations in gene expression or in cellular behavior. For example, the primary response genes induced by serum include nuclear proto-oncogenes such as c- jun (15), c-myc (16), and c-fos (17), and paracrine factors that are members of the P-thromboglobulin family including the chemotactic factors IL-8 (18) and monocyte chemotactic factor (19). With the intention of identifying primary response genes which mediate the TNF-induced activation of the endothelium, we used the technique of differential hybridization to clone eight TNF-induced cDNAs. Two of the cDNAs encoded for the cell surface receptors endothelial-leukocyte adhesion molecule-1 (ELAM-1) (20) and intercellular adhesion molecule-1 (ICAM-1) (21) that cause adhesion of leukocytes and promote their transendothelial migration. In addition, the cDNAs for two paracrine factors, IL-8 (18) and monocyte chemotactic factor (19, 21, 22), were also cloned.

Significantly, four of the cloned primary response genes were found to have no demonstratable sequence similarity to other known genes or proteins. These genes have been found to encode a novel putative zinc finger protein designated A20 (24), a novel 25-kDa secreted protein designated B61 (25), and a novel 73-kDa cellular protein designated B94.’ This diverse group of novel primary response genes has greatly expanded the field of investigation into the mechanisms by which TNF influences cellular function.

V. Sarma, F. W. Wolf, R. M. Marks, M. G. Byers, T. B. Shows, and V. M. Dixit, manuscript in preparation.

1317

1318 A Novel Primary Response Gene

In this paper we report the characterization of the fourth novel primary response gene, designated B12. B12 is investi- gated with respect to its gene structure and evolutionary conservation, induction of its transcript by proinflammatory stimuli, modulation of its transcript during development, characterization of its nucleotide and predicted protein sequence, and finally a study of the B12 protein in terms of molecular mass, expression, and half-life.

MATERIALS AND METHODS

Cell Culture and Reagents-Human umbilical vein endothelial (HUVE) cells were cultured and manipulated as described previously (26). Reagents were purchased from Sigma unless otherwise noted.

RNA and Northern Blot Analysis-HUVE cells for all experiments were used between the second and fourth passage. The cells were grown to confluence on 177-cm2 dishes (Falcon, Cockeysville MD). One day prior to treatment, HUVE cells were rinsed twice in Hanks' balanced salt solution (GIBCO) and then placed in media deficient in heparin and endothelial cell growth factor. Just prior to treatment the cells were again washed with Hanks' balanced salt solution. Cycloheximide (CHX), when used, was added 0.5 h prior to other agonists to a final concentration of 10 pg/ml. Cells were treated with recombinant human TNF (Genentech, S. San Francisco, CA) at a concentration of 20 ng/ml for the times indicated in the individual experiments. Rabbit polyclonal anti-human TNF antibody (gift of D. Remick, University of Michigan) and rabbit preimmune serum from the same rabbit were used as blocking agents. Other agonists used in this study were recombinant human IL-la (The Upjohn Co., Kala- mazoo, MI), and lipopolysaccharide (LPS, Escherichia coli Olll:B4, Sigma). Total RNA extraction and Northern blot analysis were carried out as described previously (15). Filters were hybridized with a random-primed, 32P-labeled B12 fragment (27) spanning nucleotides 872-2366 (probe B in Fig. 3A) and were washed in 2 X SSC (1 X is 0.15 M NaCI, 0.15 M sodium citrate, pH 7.0), 1% SDS at 65 "C before autoradiography.

Nuclear Run-on-HUVE cells were treated with TNF (20 ng/ml) or left untreated for 2.5 h. The cells were trypsinized from tissue culture plates and nuclei from 5 X lo7 cells were isolated as described (28). Transcripts were extended in the presence of 100 pCi [ ' Y - ~ ~ P ] UTP (3000 Ci/mmol, Amersham, Corp.), and the labeled RNA was purified as previously described (28). Linearized plasmid DNA (5 pg) was immobilized onto a nitrocellulose filter using a slot-blot apparatus (Schleicher & Schuell). The pBR322-derived plasmid pTZl8r (Phar- macia, LKB Biotechnology Inc.) served as a negative control and vimentin cDNA served as a positive control. 3 X lo6 cpm/ml of labeled transcript was hybridized to the filters for 48 h at 65 "C in a buffer containing TES (Calbiochem, San Diego, CA) pH 7.4, 0.2% sodium dodecyl sulfate (SDS), 10 mM EDTA, 0.3 M NaC1, 1 X Denhardt's solution (50 X is 1% Ficoll, 1% polyvinylpyrrolidone, and 1% bovine serum albumin), and 250 pg/ml E. coli RNA (strain B, sodium salt, Calbiochem). The filters were then washed three times at 65 "C in 2 X SSC, 0.1% SDS, and twice at 65 "C in 0.1 X SSC, 0.1% SDS before autoradiography.

Mouse Developmental Tissue Survey-Mus musculus strain CD-1 males were bred with virgin females. Embryonic day 1 was established as the day of the appearance of a vaginal plug. Embryos were removed at the indicated time points, and individual organs and tissues were excised and snap frozen in liquid nitrogen. RNA was extracted and transferred to nylon membranes (Nytran, Schleicher & Schuell) as previously described (29). Organs from littermates were pooled prior to RNA extraction and electrophoresis. A 32P-labeled B12 fragment spanning from nucleotides 154 to 1105 was hybridized to the blots at 42 "C in buffer containing 50% deionized formamide, 1 X PE (5 X PE is 250 mM Tris, pH 7.5, 0.5% sodium pyrophosphate, 5% SDS, 1% polyvinylpyrrolidone, 1% Ficoll, 25 mM EDTA, and 5% bovine serum albumin), 5 X SSC, and 150 pg/ml salmon sperm DNA. The blots were washed at 65 "C in 2 X SSC, 1% SDS, and subjected to autoradiography for 1 week at -80 "C.

Library Construction and Screening-Construction and differential screening of the oligo(dT)-primed TNF-induced, CHX-treated HUVE cDNA library was described previously (26). Full-length cDNAs were obtained by further screening of an unstimulated HUVE cell random-primed library (courtesy of D. Ginsburg, University of Michigan), and a TNF-induced, CHX-treated HUVE cell random- primed library with 32P-labeled probes corresponding to the B12.0 insert (see Fig. 3A) and a 580-base pair EcoRI fragment from the 5'

end of the EC2.1 clone (probe A in Fig. 3A). Hybridizing plaques were purified and their cDNA insert subcloned into the pGEM7zf vector (Promega Corp., Madison, WI) by standard methods (29).

Sequencing and Sequence Analysis-Plasmid DNA purified by cesium chloride banding (29) was sequenced on both strands using the dideoxynucleotide chain termination method with modified T7 DNA polymerase (30) and synthetic oligonucleotides as primers. Where necessary sequence compressions due to template secondary structure were resolved using the deaza base analog of guanosine. Sequence data was compiled and analyzed using the Genetic Com- puter Group's Sequence Analysis Software (v6.2) (31). Databases searched via the Genbank electronic mail service were Genbank (release 67.0) and EMBL (release 25.0) DNA databases, and Swiss- Prot (release 17.0) and the translated Genbank (release 64.3) protein databases using the FASTA algorithm (32). These and other databases were likewise searched using the BLAST algorithm (33, 34) (courtesy of M. Boguski, National Center for Biotechnology Infor- mation, National Institutes of Health, Bethesda, MD).

Primer Extension-High pressure liquid chromatography-purified synthetic oligonucleotides complementary to nucleotides 42-71 and 93-122 of the B12 cDNA were 5' end-labeled with [-p3'P]ATP (ICN Biomedicals, Irvine, CA) and T4 polynucleotide kinase (Boehringer Mannheim) to a specific activity of 5 X lo6 cpm/pmol and the labeled primers were purified on Nensorb columns (Du Pont-New England Nuclear). One pmol of labeled primer was annealed to 30 pg of TNF- stimulated, CHX-treated HUVE cell total RNA or to 30 pg of tRNA in 5 mM phosphate buffer, pH 6.75), containing 5 mM EDTA. The primer-template mixture was heated to 90 "C, and NaCl was added to a 10 mM final concentration. After slow-cooling the mixture to 30 "C, primer extension was carried out for 75 min using avian myeloblastosis virus reverse transcriptase (Seikagaku, Rockville, MD) in the presence of 10 mM dithiothreitol, 60 units of RNasin (Promega), 50 mM Tris-HC1, pH 8.3, 5 mM MgC12, and 1 mM deoxy- nucleoside triphosphates (Pharmacia). The extended products were phenol/chloroform (25:24) extracted, ethanol precipitated, and resuspended in sequencing termination solution (United States Biochem- icals), and resolved on an 8 M urea, 6% polyacrylamide sequencing gel.

Genomic DNA Analysb-Human genomic DNA was extracted from the 11B squamous carcinoma cell line as described (29). DNA was digested with the indicated restriction endonucleases, electropho- resed through 0.6% agarose gels, and transferred to nitrocellulose (Schleicher & Schuell) by capillary transfer. Genomic DNA from other species (gifts of P. Killen, University of Michigan, and P. Venta, Michigan State University) were digested with BglII and transferred to membranes as above. The blots were hybridized to 32P- labeled B12 probe (probe A, Fig. 3A) at 42 "C overnight in 30% formamide, 5 X SSC, 5 X Denhardt's solution, 0.1% SDS, and 100 pg/ml salmon sperm DNA. The filters were washed from conditions of low stringency (2 X SSC, 0.1% SDS at room temperature) to conditions of high stringency (0.1 X SSC, 0.1% SDS at 68 "C) before autoradiography.

Chromosomal Localization-Metaphase chromosome spreads of synchronized normal peripheral blood lymphocyte cultures were pre- pared as previously described (35, 36). A 1.6-kilobase B12 probe corresponding to nucleotides 872-2484 was biotinylated and hybridized to the metaphase chromosome slides. The hybridized probe was then detected using fluorescein avidin DCS (Vector Laboratories, Burlingame, CA). Chromosomes were counterstained with propidium iodide and 4',6-diamidino-2-phenylindole, and observed with a Nikon fluorescence microscope.

Fusion Protein Production-A 580-base pair EcoRI fragment corresponding to nucleotides 292-872 purified from the B12 EC2.1 clone was ligated into the EcoRI site in the polylinker of the E. coli trpE fusion protein vector pATH22 (37, 38). The recombinant expression plasmid transformed into E. coli DH5a cells was sequenced across the vector/insert boundaries to determinme insert orientation and to confirm that the reading frame was maintained. Transformed cells were grown to an optical density at 600 nm (ODW) of 0.3-0.6 in 10 ml of M9 media (29) including 1% casamino acids (Difco Laboratories, Detroit, MI), 1 mM MgSO,, 0.1 mM CaCl', 0.2% glucose, 20 pg/ml thiamine, 150 pg/ml ampicillin, and 20 pg/ml L-tryptophan. The cells were pelleted and then resuspended in 10 ml of the above media without tryptophan. One ml of this culture was used to inoculate 100 ml of the above media also lacking tryptophan. After 1 h of incubation at 37 "C the artificial inducer of the trpE operon 3P-indoleacrylic acid, dissolved to 10 mg/ml in 100% ethanol, was added to a 10 pg/ ml final concentration, and the culture was allowed to grow an

A Novel Primary Response Gene 1319

additional 4-6 h at 37 “C. Bacterial cells were pelleted, lysed with lysozyme on ice, and sonicated. To purify the insoluble fraction containing fusion protein the resulting bacterial lysate was pelleted and resuspended sequentially in 20 ml of 0.3 M NaCl with 0.5% Nonidet P-40, 20 ml of 0.1 M NaCl with 10 mM Tris, pH 7.4, and 20 ml of 10 mM Tris, pH 7.4. The final pellet was resuspended in 1 ml of 10 mM Tris, pH 7.4, and aliquots of the fusion protein were stored a t -20 “C.

A second B12 fusion protein was created in the PET expression system (39), in which exogenous DNA sequences are expressed as a fusion product with the gene 10 protein under control of the T7 promoter. The polymerase chain reaction (PCR) was used to amplify a 659-base pair fragment of the B12 EC2.1 clone using the primers

CCTGGGAATTCCAC-3’, the latter introducing a BamHI site. This fragment was digested with BamHI and cloned into pET3xc. Plasmid purified from transformants was digested with BgZII to determine insert orientation, and one of the expression plasmids containing insert in the correct orientation was fully sequenced to ensure fidelity of the PCR amplification. The plasmid construct was then transformed into E. coli strain BL21(DE3)pLysS, which contains the T7 polymerase gene under control of the inducible lacUV5 promoter. The cells were grown at 37 “C in 10 ml of M9 medium containing 1% NZ Amine A (GIBCO), and 100 pg/ml ampicillin to an OD,, of 0.6. Isopropyl-P-D-thiogalactopyranoside was added to a final concentration of 1 mM and the culture was grown for an additional 3 h. The cells were pelleted, resuspended in 1 ml of 2 mg/ml lysozyme dissolved in phosphate-buffered saline, pH 7.4, freeze-thawed twice, and then sonicated three times on ice after the addition of 0.5% Nonidet P-40. The inclusion body fraction of the lysate was pelleted by centrifuga- tion at 15,000 X gin a JS13.1 rotor for 2 min at 4 “C, and was washed twice in 1 M urea dissolved in phosphate-buffered saline, pH 7.4, at room temperature. The final pellet was stored at -20 “C suspended in phosphate-buffered saline, pH 7.4.

Approximately 100 pg of insoluble pATH22/B12 fusion protein and approximately 100 pg of insoluble pATH22 fusion protein backbone were solubilized by the method of Marston (40). The fusion protein-containing supernatant was concentrated 10-fold by centrif- ugation through centricon-30 columns (Amicon, Danvers, MA) at 5000 X g, and aliquots were stored at -20 “C. Presence of cognate fusion protein was verified by immunoblot analysis.

Polyclonal Antiserum-pATH22/B12 fusion protein (500 pg) solubilized in SDS sample buffer (39) was resolved by electrophoresis through a 10% SDS-polyacrylamide gel. After light staining with Coomassie Blue and multiple washes with H20 to remove SDS, the area of the gel containing fusion protein was excised and emulsified in complete, and subsequently incomplete Freund’s adjuvant. Two female New Zealand White rabbits were injected intramuscularly at 3-week intervals and antisera was collected 2 weeks following each injection. Antisera specificity was determined by immunoblotting (41). Briefly, the purified fusion proteins and their corresponding backbone proteins were separated on 10% SDS-polyacylamide gels, and transferred to nitrocellulose membranes using a semi-dry electro- blotting apparatus (LKB Multiphor, Pharmacia) according to instructions supplied by the manufacturer. The membranes were blocked overnight with 3% non-fat dry milk in Tris-buffered saline, pH 7.4, and then incubated with immune or preimmune serum diluted from 1:lOO to 1:20,000. Bound antibody was detected using a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody in con- junction with the color development reagent 4-chloro-1-naphthol (Bio-Rad).

In Vitro Transcription and Translation-The open reading frame of B12 was selectively amplified away from both 5’- and 3”untrans-

AGCTTGCCATGGCGGGGGACACCTGCCTG-3’ and 5”CCTGA- lated regions of the B12 cDNA by PCR. The two primers 5”CCTGA-

CAAGCTTAGGCCATGGTCAGTCGCGATGG-3’ include engi- neered HindIII sites 8 base pairs upstream of the initiator methionine codon and 10 base pairs downstream of the stop codon. The sense strand primer also places the initiator methionine in a more favorable context for translation initiation (42). DNA isolated from the TNF and CHX-treated, oligo(dT)-primed HUVE cell cDNA library (26) was used as template for PCR (43). The resulting 976-base pair fragment was digested with HindIII and cloned into pGEM7zf (Pro- mega), and the entire insert was sequenced to ensure PCR fidelity. This construct was linearized with MluI and was used as template for T7 RNA polymerase as per the instructions of the supplier (Promega). The resulting transcript was phenol/chloroform extracted and ethanol precipitated. One-seventh of the total transcript was used

5”AGTATGTCCAGCTCAACGTG-3’ and 5”CATAGATI’CGGATC-

to program a wheat germ extract (Promega) in a reaction including 25 pCi of ~- [~~S]cys te ine (Amersham Corp.) and carried out according to the manufacturers’ suggestions. Immunoprecipitation of the in vitro translated material was essentially as described (44). For blocking experiments, antisera was incubated 2 h a t room temperature with approximately 2.5 pg of solubilized pATH22/B12 fusion protein or pATH22 backbone protein in 1 ml of phosphate-buffered saline containing 1% Triton X-100 0.5% deoxycholic acid and 0.1% SDS and 1 X protease inhibitor mixture (1000 X is 1 mg/ml leupeptin, 1 mg/ml aprotinin, 10 mg/ml soybean trypsin inhibitor, and 1 mg/ml pepstatin).

Metabolic Labeling and Immunoprecipiuttion-Second passage HUVE cells grown to confluence on fibronectin-coated 90-mm dishes were deprived of growth factors for 16 h prior to stimulation. The cells were washed twice with EMEM (Whittaker Bioproducts, Walk- ersville, MD) and placed in EMEM containing 5% filtered, dialyzed, low endotoxin ( ~ 0 . 1 unit/ml) fetal calf serum (Hyclone, Logan, UT), and 20 ng/ml TNF for 6 h. After washing the cells twice, they were placed for 30 min in media deficient in cysteine and methionine but including a mixture of [35S]cysteine and [35S]methionine (Tran3’S- label, ICN) at 400 pCi/ml and 20 ng/ml of TNF. Conditioned media and cell layer fractions were then collected and equivalent numbers of trichloroacetic acid precipitable counts were immunoprecipitated as previously described (44). RNA was purified from HUVE cells treated identically except that after 6 h of TNF stimulation the cells were placed into EMEM containing 5% fetal calf serum and 20 ng/ ml TNF for 30 min. Relative band intensities were quantitated using the Image program (version 1.35) from National Institutes of Health on an Apple Macintosh computer.

For determination of the rate of B12 protein degradation, HUVE cells deprived of growth factors overnight were washed twice and placed in cysteine-free and methionine-free EMEM containing 0.1% bovine serum albumin (low endotoxin, Sigma) for 30 min. Recombi- nant human TNF (20 ng/ml) and [35S]cysteine and [35S]methionine (100 pCi/ml, Amersham Corp.) were added and the cells were incubated for 7 h. Label was removed from the media, and the cells were harvested either without a chase period or after 0.5, 2, and 4 h of chase and immunoprecipitated.

RESULTS

Expression of B12 Transcript-One of the TNF-induced cDNAs cloned by differential hybridization (26) was designated B12. The 1.5-kilobase insert of the B12 clone was used to probe RNA blots derived from HUVE cells that were either untreated or treated with TNF and/or CHX (Fig. U). A transcript of approximately 3.5 kilobases was detected which was variably present at low levels in untreated cells (lane 18). Transcript began to accumulate within 15 min of treatment (lane 2 ) , and continued to increase until 4 h of treatment (lanes 5, 8, 11, and 14) after which message levels declined (lane 17). Treatment with CHX and TNF together (lanes 4, 7, 10, 13, and 16) enhanced rather than blocked message accumulation, demonstrating that protein synthesis is not required for B12 induction. Cycloheximide treatment alone also induced B12 message (lanes 1, 3, 6, 9, 12, and 15), although more transiently than TNF (compare lanes 15 and 17). The blot was hybridized with a probe for vimentin, an intermediate filament protein whose message is expressed constitutively at high levels in HUVE cells and is not respon- sive to TNF treatment, to ensure equivalent loading of RNA samples.

Other proinflammatory agents were tested for their ability to induce B12 transcript in HUVE cells (Fig. 3B) . Treatment of HUVE cells for 2 h with IL-lP resulted in induction of B12 message (lane 5 ) , and marginal superinduction in the concom- itant presence of CHX (lane 6 ) . LPS similarly induced the B12 transcript (lanes 7 and 8). To eliminate the possibility of LPS contamination as being the cause of B12 induction, HUVE cells were treated with TNF in the presence of polyclonal antisera directed against TNF. B12 message was barely detectable in the presence of anti-TNF antisera (lane 9) but was comparable to TNF stimulation (lane 5 ) in the presence


1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8

1 2 3 4 5 6 7 8 9 1 0

FIG. 1. Time course of induction of B12 transcript by TNF ( A ) , induction b y other proinflammatory cytokines ( B ) , and nuclear run-on analysis (C). A, HUVE cells were treated for times indicated above the blot with TNF (20 ng/ml) and/or CHX (10 pg/ ml) and total RNA was extracted, resolved (10 pgllane) through a formaldehyde-agarose gel, electroblotted onto nylon membrane, and probed with "P-labeled B12 cDNA (fragment B in Fig. 3A) or vimentin to ensure equivalent RNA loading. B, cells were treated for 2 h with T N F (20 ng/ml), CHX (IO pglml), IL-lP (5 units/ml), LPS (1 pg/ml), rabbit anti-human T N F antibody (Anti-TNF Ab), whole serum diluted 1:100, and preimmune serum (Control Ab) from the T N F antibody donor, whole serum diluted 1:lOO. RNA was processed as in A. C. Nuclei from HUVE cells that were either unstimulated or stimulated with T N F (20 ng/ml) for 2.5 h were harvested and used for run-on transcript synthesis as described under "Materials and Methods." These transcripts were then hybridized to the linearized plasmid DNAs indicated to the left of the filters.

of preimmune serum (lane 10). Again vimentin was used as a control for equivalent loading of RNA samples.

Nuclear Run-on-In order to determine the mechanism of TNF-induced B12 transcript accumulation, run-on transcription of untreated and TNF-treated (2.5 h at 20 ng/ml) nuclei was performed. Radiolabeled nascent RNA from the nuclei was hybridized to linearized plasmid DNA immobilized on nitrocellulose filters (Fig. 1C). The plasmid backbone pTZ18r (a pBR322 derivative) was used as a negative control and showed no hybridization. Plasmid-containing vimentin gave equivalent hybridization in untreated and treated cells demonstrating that the specific activities of the radiolabeled RNAs were equivalent. The signal for B12 appeared to be induced at least 2-fold over basal level transcription, suggesting that transcriptional activation contributes to the induction of B12 mRNA in response to TNF.

Developmental Regulation of the B12 Transcript-The developmental expression of B12 transcript was determined by Northern blot analysis of RNA extracted from various tissues of the mouse embryo (Fig. 2). B12 message was detectable in the embryonic liver 15 days post-gestation (lane 1 ) after which it rapidly became undetectable (lanes 2-6). In contrast, B12 message in kidney tissue increased to a peak expression on the day of birth (lanes 7-9), after which transcript levels declined (lanes 10 and 11 ). B12 message was first seen in the heart on day 17 (lanes 12 and 13) and persisted through adulthood (lanes 14-1 7). Conversely, B12 message in the lung was present at the earliest developmental stage examined (lane 18) and persisted through to the day of birth, after which it became undetectable (lams 19-23). In the placenta B12 message was found to be expressed constitutively from day 13 through day 17, and in the brain from embryonic day 15 through adulthood (data not shown). Ethidium bromide-

stained RNA transfers are shown to demonstrate the relative equivalency of RNA loading.

Characterization of the B12 cDNA and Predicted Protein- A schematic representation of the B12 cDNA and the cDNA fragments obtained through library screening is presented in Fig. 3A. Clone B12.0 was derived from the differentially screened HUVE cell cDNA library. This clone was used as a probe to rescreen both the original library as well as an unstimulated endothelial cell cDNA library, resulting in the isolation of three clones: B12A from the TNF- and CHX- treated library, and EC2.1 and E90 from the unstimulated library. The relatedness of these clones was established through restriction analysis, Southern blot cross-hybridization, and sequence analysis. As the derived consensus sequence did not contain a plausible initiator methionine, a third round of screening was undertaken, using a 580-bp EcoRI fragment (probe A, Fig. 3A) of the EC2.1 clone as a probe for a random-primed, TNF- and CHX-treated HUVE cell cDNA library. Three cDNAs containing sequence 5' to the EC2.1 cDNA were cloned and characterized. The longest, designated B12E, is represented in Fig. 3A. The two other clones provided evidence that the 5' region of the deduced B12 cDNA was authentic, since one of these clones terminated only 8 bp downstream of the 5' end of the clone B12E.

The nucleotide and deduced protein sequence of B12 is shown in Fig. 3B. The composite 3512-bp cDNA sequence has a 153-bp 5"untranslated region, a continuous open reading frame of 951 bp, and a long 3"untranslated region of 2408 bp. The 5'-untranslated region has a 75% G+C content in the first 50 bases and contains stop codons in all three reading frames, precluding the existence of an initiator methionine upstream of the cloned sequence. Within the 3"untranslated sequence there exist two consensus polyadenylation signals and one putative message destabilizition signal, the latter of which is a common element in cytokine transcripts and in primary response genes (45, 46). The first ATG in from the 5' end of the sequence is located at base 154 in a context favorable to translation initiation (42). Translation of the open reading frame predicts a 316 amino acid polypeptide with a predicted molecular mass of 36.2 kDa. Recently, there has been reported evidence for translational initiation from non-AUG codons upstream of the presumed initiator methionine codon (47,48). A single in-frame CTG found 32 codons upstream of the ATG is found in a context favoring translation initiation (42) and would extend the mature polypeptide to 348 amino acids predicting a molecular mass of 39.6 kDa. Charged residues comprise 29% of the 316-amino acid predicted polypeptide, with a predominance of arginine, lysine, and asparagine residues. Indeed, 20 of the 36 carboxyl-terminal residues are charged.

Extensive database searches with B12 at both the DNA and protein level failed to reveal any similarities with known sequences. Further searches against the PROSITE protein motif data base (release 6.10) with the B12 protein were negative except for highlighting a potential N-linked glyco- sylation site at residues 163-166. Comparison of the protein to itself revealed one weak internal similarity between residues 207-219 and residues 222-234 with the consensus of Glu- X-Cys-Cys-X-Ser-X(I-2,-Tyr-X(4,-Lys, where X is any amino acid. No other sequence in the protein databases searched had this consensus sequence.

Two regions of the protein sequence have residue and charge distributions similar to elements found in other proteins: first, a single region with the potential of forming an amphipathic helix extends from amino acid 177 to amino acid 194. This potential helical region would be stabilized by salt


FIG. 2. B12 transcript is differ- Liver Kidney Heart Lung entially expressed in the developing i

mouse embryo. Tissues were harvested I w w w m m '"w IJJ m m 5IIw w w P P 'I" w w P P 5 from mouse embrvos 15.17. and 19 davs ~ ~ ~ - ~ a ~ ~ - ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ - ~ ~

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post-gestation ( k n e s j5E, 17E, and ~ 8 ~ - 19E) , from newborn mice the day of and the day after birth (lanes Day I and Day Z ) , and from adult mice. RNA (10 pg) from the tissues indicated ahove the blot was fractionated by electrophoresis through a formaldehyde-agarose gel, 18s -

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fragment representing the entire reading frame. Upper panel is an autoradiograph of the filters after a high stringency wash. Lower panel is photographs of ethidium bromide-stained RNA follow- I

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R3

116

1 4 9

1 8 3

216

7 4 9

2 8 3

31 6

FIG. 3. Schematic map and sequencing strategy of B12 cDNA clones ( A ) , the BIZ nucleotide and derived amino acid sequence ( B ) , and the amino acid hydropathic profile (C). A, five representative clones and a composite B12 cDNA are shown schematically. Restriction sites, both unique and relevant to cloning, are shown along the composite sequence. The 5'-untranslated region, possible CTG initiation codon, AUG initiator methionine, open reading frame, and 3'- untranslated region are shown by differential shading across the clone maps. Numbers indicate nucleotide base number. Probes A and B were used for South- ern and Northern analysis, respectively. Arrows below clones indicate the extent of sequencing done for each clone. R, nucleotide numbering is on the left, and amino acid numbering is on the right of the figure. Amino acid numbering begins with the first in-frame AUG a t nucleotide 154, and a putative alternate CUG initiation codon and open reading frame of 32 amino acids upstream of the AUG initiator methionine is shown in italics and in parentheses. The stop codon is indicated with an asterisk and stop codons in all three frames upstream from the putative initiator codon are shaded. Two polyadenylation signals in the 3'- untranslated region are underlined. An ATTTA message destabilization signal at base pair 3070 is indicated by a dashed underline. Oligonucleotides used for primer extension analysis (Fig. 4) are numbered and shown as arrows above the sequence. C, hydropathic profile for the open reading frame starting at amino acid -32 was generated using the Kyte and Doolittle algorithm (51) with a window setting of 8. Regions of hydrophil- icity are shown below the centerline.


bridges formed by oppositely charged amino acids found along the charged face (49). Amphipathic helices are found in phos- pholipid-interacting proteins (50), and in helix-loop-helix DNA-binding proteins (50). Second, residues 274-289 have the characteristic charge distribution and amino acid content of a PEST protein sequence (19). Proteins classified as PEST have short regions of sequence rich in the amino acids proline, glutamate, serine, and threonine (hence the acronym PEST) which are bounded by basic residues. Most rapidly proteolysed proteins contain a PEST region, although the converse is not necessarily true.

The B12 protein sequence hydropathic profile is shown in Fig. 3C. The plot was generated using the Kyte and Doolittle (51) algorithm with a moving window of 8 residues. Of signif- icance is the lack of any lengthy hydrophobic stretches, often indicative of membrane-spanning proteins, and the lack of a hydrophobic signal sequence. The carboxyl terminus of the predicted polypeptide is shown as being very hydrophilic which is in keeping with the charge characteristics outlined above.

Primer Extension-In order to determine the 5' terminus of the B12 transcript, primer extension analysis was performed. Using 30 pg of RNA from TNF-stimulated HUVE cells as a template, end-labeled oligonucleotide primers com- plimentary to the 5' end of B12 (see Fig. 3B) were extended using AMV-reverse transcriptase. The single-stranded cDNA products derived from extending primer 1 are shown in Fig. 4. Although many products are apparent in the extension reaction ( l u n e 2), only those marked with arrows at the left of the figure were present in both reactions. Bands are evident at 4-7 base pairs and 55-56 base pairs 5' to the end of the cloned cDNA. Alternate transcription initiation sites could account for the occurrence of these two reaction products. Since there are stop codons in all three frames in the sequenced 5' end of the molecule, extension of the reading frame into the uncloned region is impossible. tRNA was used as a template in a parallel reaction to establish background priming in the analysis ( l a n e 3).

Genomic Southern Analysis-DNA extracted from the 11B squamous cell carcinoma cell line was digested with the restriction enzymes indicated in Fig. 5A and fractionated by electrophoresis through a 0.6% agarose gel. The DNA was transferred onto a nitrocellulose membrane and probed with a "P-radiolabeled EcoRI cDNA fragment which represented about 60% of the B12 coding region (probe A in Fig. 3A). Washes were performed ranging from low stringency (2 x

1 2 3

-55"

-7 - -4 "

FIG. 4. Primer extension analysis. Total RNA from HUVE cells stimulated with TNF (20 ng/ml) was used as template with the oligonucleotides shown in Fig. 3B as primers. Lane 1 is a T lane sequencing ladder; lane 2 is a primer extension using oligonucleotide 1; lane 3 is control primer extension using oligonucleotide 1 as primer and tRNA as template. Numbers at left indicate length of extended product in bases beyond the reported 5' end of the B12 cDNA shown in Fig. 3B.

12.2- kb

9.2-

7.1 - 5.1 - 4.1 -

3.1 -

2.0 -

1.0-

FIG. 5. Copy number ( A ) and evolutionary conservation ( B ) of the B12 gene. A, human DNA (12 pg) was digested with the restriction enzymes indicated above the blot, and processed as described under "Materials and Methods." The blot shown was washed in 0.1 X SSC at 65 "C for 20 min. Molecular size standards are shown to the left of the blot. B, human, macaque, rat, mouse, rabbit, and Drosophila DNA was digested with the restriction enzyme BgnI and processed as described under "Materials and Methods." The blot shown was washed in 0.1 X SSC a t 68 "C for 20 min. Molecular size standards are indicated to the left of the blot.

SSC at 40 "C) to high stringency (0.1 X SSC at 68 "C) in a stepwise manner. Shown in Fig. 5A is the Southern blot after the most stringent wash. All hybridizing bands present at the less stringent washing conditions persisted through the more stringent conditions, which is consistent with B12 not being part of a sequence related gene family. Additionally, the appearance of one discrete band in the EcoRI and SstI digests indicates that B12 exists as a compact single copy gene in the human genome.

To determine the evolutionary conservation of the B12 gene, genomic DNA from human, macaque, rat, mouse, rabbit, and Drosophila were digested with BgIII, fractionated by electrophoresis through a 0.6% agarose gel, and transferred to a nitrocellulose membrane. The filter was probed with a "P-radiolabeled B12 fragment (probe A in Fig. 3A) and then washed in 0.1 X SSC at 65 "C. The resulting autoradiograph is shown in Fig. 5B where strongly hybridizing bands are detectable in all species including Drosophila. Of note is the roughly equivalent size of hybridizing bands in the human and macaque and in the rat and mouse DNA digests, also evident in these DNAs when digested with EcoRI, suggesting a high degree of evolutionary conservation of the B12 gene.

Chromosomal Localization of the B12 Gene"B12 was localized to the long arm of human chromosome 17 in the q22 + q23 region by in situ hybridization (Fig. 6A and B). A total of 50 metaphase spreads with a total of 94 fluorescent signals were analyzed. 35% of the total number of signals were localized to chromosome 17 (Fig. 6A), and 54.5% of the signals on chromosome 17 were located at bands 17q22 + q23 (Fig. 6B). No more than two fluorescent signals were apparent at any one location on chromosomes other than 17. The B12 gene ( TNFAIPl ) has also been mapped to chromosome 11 in the mouse by intersubspecific backcross analysis (edp-1) (52). Human chromosome 17 and mouse chromosome 11 share large regions of homology, and thus these two mapping studies are in agreement. Interestingly, edp-1 mapped in the mouse to less than 1cM distal to the tumor supressor gene p53 and


A B FIG. 6. The B12 gene is on human

chromosome 17. Peripheral blood lym- 0.. phocyte chromosome spreads were hybridized with a 1.6-kb biotinylated B12 probe and counterstained with propi- ,o dium iodide and 4',6-diamidino-2-phen- .c ylindole. Fifty metaphase spreads were 5 analyzed. A, distribution of fluorescent E signals on the chromosomes. Number of E signals in one area is shown vertically 2 30

and chromosomes are depicted horizon- ........ tally. B, ideogram of chromosome 17 IO .......... showing fluorescent signal distribution. o 0.. 54.5% of the signals on chromosome 17 were located on bands 17q22-23.

0 0

0 0

0..

0. Chromosomes

17 the gene for the RNA polymerase I1 large subunit, both of which are localized to 17~13.1 in humans (53). This suggests that a chromosomal rearrangement in the area between these genes has occurred in humans when compared to mice. Distal to the edp-1 gene on the mouse chromosome is the eui-2 locus, which is localized to 17qll+ q12 in the NF1 region of human chromosome 17 (54, 55), suggesting that a further rearrangement event occurred just distal to the edp-1 gene.

Generation of B12 Polyclonal Antiserum-Rabbit polyclonal antisera directed against B12 was raised using a bacterially expressed B12 fusion protein as immunogen. A fragment of the B12 cDNA which includes 192 amino acids of the open reading frame was fused in frame to the bacterial trpE protein encoded in the PATH vector system (Fig. 7, A and B ) and was subsequently expressed at high levels in bacteria (Fig. 7C, left panel). This approximately 55-kDa fusion protein was used to immunize rabbits.

To generate a reagent to test for antibodies specific to B12 a 172 amino acid fragment similar to that expressed in the PATH system was fused to the gene 10 protein using the PET vector system (Fig. 7, A and B ) and was similarily expressed to high levels (Fig. 7C, left panel). The presence of antibodies specific to B12 was determined by immunoblotting (Fig. 7C, right panel). As expected, the polyclonal immune serum detected both the pATH22/B12 fusion protein and the pATH22 trpE backbone alone, since the rabbits were immunized with the pATH22/B12 fusion protein which contains both B12 and backbone sequences. The antiserum stained the pET3xc/ B12 fusion protein but not the gene 10 backbone, confirming the presence of anti-B12 antibodies in the antiserum. Incu- bation of an identical blot with preimmune serum did not result in any staining (data not shown).

In Vitro Translation of in Vitro Transcribed B12 mRNA- In order to demonstrate that the B12 transcript directs the synthesis of a protein of the predicted molecular mass (36 kDa), the B12 cDNA open reading frame was cloned downstream of a T7 promoter and transcribed in vitro using T7 RNA polymerase. The resulting transcript was as expected approximately 1 kilobase in length. This transcript directed the synthesis of a 36-kDa polypeptide in a wheat germ extract (Fig. 8, lane 1 ). Preimmune serum did not immunoprecipitate any radiolabeled proteins from the extract (lane 2). The B12 polyclonal antisera specifically immunoprecipitated the 36- kDa translation product (lane 3), and this interaction was blocked by preincubating the antisera with soluble pATH22/ B12 fusion protein (lane 4 ) , but was not blocked by preincubating the antisera with soluble pATH22 backbone protein alone (lane 5 ) . These data demonstrate that the B12 gene is able to direct the synthesis of a 36-kDa polypeptide, which is

FIG. 7. Generation of polyclonal antiserum. A, schematic diagram of the B12 cDNA. Upper line represents the B12 cDNA with features outlined as described in Fig. 3A. The fragment used for PATH expression extends from an EcoRI site (inparentheses) derived from the linker used in library construction in clone EC2.1 to the internal EcoRI site a t base 872. The B12 cDNA fragment used in the PET expression system extends from the internal BarnHI site at base pair 363 to a BarnHI site articially introduced 8 base pairs downstream of the internal EcoRI site. B, schematic diagram of the constructs used in antibody production and characterization. Left panel, the B12 EcoRI fragment is shown cloned into the EcoRI site of the pATH22 vector. BarnHI sites used in determining the orientation of the insert are also shown. The location of the trpE gene and the number of amino acids of trpE expressed in the fusion product are shown. Right panel, the BarnHI fragment of B12 is shown cloned into the pET3xc vector, such that 260 amino acids of the gene 10 prot.ein are fused in frame to 172 amino acids of B12. The location of the BgnI sites used for determining insert orientation are shown, as well as other sites in the cloning region. Antibiotic resistance genes and plasmid origin of replication sites are depicted for both constructs. C, expression of B12 fusion proteins in E. coli and generation of anti-B12 antibodies. Molecular mass markers are indicated in kilodaltons to the left of each blot. Left panel, Coomassie stained 10% SDS-polyacrylamide gel showing fusion protein expression. Lanes from left to right are: unin- duced pATH22/B12 construct-containing bacterial cell lysate (Un- induced culture), induced pATH22 vector cell lysate (pATH22), induced pATH22/B12 cell lysate insoluble fraction (pATH22/B12), induced pET3xc vector cell lysate (pET3xc), and induced pETBxc/ B12 cell lysate-insoluble fraction (pET3xclBlZ). Right panel, immunoblot of the fusion proteins and fusion protein backbones stained with rabbit anti-B12 antiserum diluted 1:10,000. The protein samples used for immunoblotting are the same as those shown in the left panel of this figure.

in agreement with the sequence data, and that the anti-B12 polyclonal antisera recognizes this polypeptide.

Expression of the B12 Protein in Endothelial Cells-To characterize the cellular location and the TNF responsiveness of the B12 protein, HUVE cells were metabolically labeled in the presence or absence of TNF, and the conditioned media


@ 1 2 3 4 5 @ 6 7 8 9 10 0 1 1 1 2 1 3 1 4

66 - - kDa

45 - 6 -

5 - p

FIG. 8. Characterization of the B12 protein. A, the B12 gene encodes a 36-kilodalton polypeptide. Wheat germ cell lysates were programed with in vitro transcribed B12 as described under “Mate- rials and Methods.” One-seventh of the resulting radiolabeled protein was immunoprecipitated and fractionated on a 10% SDS-polyacrylamide gel. Molecular mass standards are indicated to the left of the blot. Shown is the programed cell lysate before immunoprecipitation (lane I ) , and immunoprecipitation of the lysate with preimmune serum (lane 2), immune serum (lane 3), immune serum preincubated with soluble pATH22/B12 fusion protein (lane 4 ) , and immune serum preincubated with soluble pATH22 protein backbone (lane 5). Preim- mune and immune serum were used at a 1: lOO dilution. B, B12 protein expression in HUVE cells. Cells were metabolically labeled in the presence or absence of TNF as described under “Materials and Methods.” RNA was extracted from cells treated in parallel. All lanes were immunoprecipitated with the B12 antibody except lane 6 which was immunoprecipitated with B12 antibody that had been preincubated with soluble pATH22/B12 fusion protein. Lanes 6 and 8 are immunoprecipitations of the cell layer treated with TNF for 6.5 h. Lane 7 is an immunoprecipitation of untreated cell layer. Lanes 9 and 10 are immunoprecipitations of the conditioned media from HUVE cells not treated or treated with TNF for 6.5 h, respectively. Molecular mass standards are indicated at the left of the blot in kilodaltons. Below lanes 7 and 8 is a Northern blot of HUVE cell total RNA treated with TNF for the times indicated below the blot. To the right of each panel the probe is specified. C, the B12 protein is rapidly degraded in TNF-treated HUVE cells. HUVE cells were metabolically labeled for 7 h in the presence of TNF. Radioactive amino acids were then removed and the cell layer harvested immediately (lane 11 ), after 30 min (lane 12), after 2 h (lane 13), or after 4 h (lane 1 4 ) of incubation in fresh medium containing unlabeled amino acids. Mo- lecular mass standards are indicated to the left of the blot.

and cell layer of the treated cells immunoprecipitated with anti-B12 polyclonal antisera. B12 protein was detected in the cell layer as a 36-kDa band (lane 7), but not in the conditioned media (lane 9) from cultured HUVE cells, indicating that B12 is not a secreted protein. Stimulation with TNF for 6.5 h led to a doubling in B12 protein expression (compare lanes 7 and 8) without any detectable accumulation of B12 protein in the media (lane IO). B12 was not detectable in the conditioned media as late as 16 h after TNF induction under chronic labeling conditions (data not shown). Preincubating the B12 antisera with soluble pATH22/B12 fusion protein blocked the immunoprecipitation of the 36-kDa band in the cell layer (lane 6).

To correlate B12 protein induction with induction of its transcript, RNA samples were extracted from HUVE cells treated identically to those used for immunoprecipitation analysis. In this particular experiment B12 transcript was found to be induced 2.5-fold (shown below Fig. 8B) which correlates with the doubling in level of B12 protein. Thus even though the degree of induction of B12 transcript is highly variable from experiment to experiment (compare Fig. lA,

lane 8, and the Northern blot in Fig. 8B) and may depend on undefined parameters such as cell cycle status or degree of confluence, this experiment nevertheless demonstrates that the induction of B12 transcript is mirrored in the induction of B12 protein, making translational or post-translational control unlikely.

B12 protein half-life was measured by immunoprecipitation of HUVE cell layer samples from cells that had been metabolically labeled with [3sS]methionine and [”’Slcysteine in the presence of TNF for 7 h and then chased with label-free media containing TNF for varying lengths of time. Shown in Fig. 8C are samples immunoprecipitated after no chase period (lane 11), after 0.5 h (lane IZ), after 2 h (lune 13), and after 4 h of chase (lane 14). It is apparent that at least half of the B12 protein has been degraded before 2 h, possibly as early as 0.5 h. Samples treated in the same manner as above but in the absence of TNF stimulation gave similar results (data not shown) demonstrating that the rate of B12 protein degradation is not detectably affected by TNF stimulation. The pulse- chase study confirms that B12 is a protein with a short half- life as it is rapidly degraded.

DISCUSSION

The initial transcriptional response of HUVE cells to proinflammatory stimuli has been found to involve a surprisingly large number of genes which orchestrate the dramatic phe- notypic alterations in endothelium that are necessary for the development of an inflammatory response. Primary response genes in HUVE cells include those responsible for the recruit- ment (e.g. IL-8 and monocyte chemotactic factor) and adhesion of leukocytes (e.g. ELAM-1, ICAM-1, and vascular cell adhesion molecule-1 (56)), those which establish the proco- agulant phenotype (e.g. plasminogen activator inhibitor-1), and those that modulate transcription (e.g. c-jun). We have additionally cloned by differential hybridization a set of four novel primary response genes, including B12, that likely contribute to one or more of the above listed effects of TNF.

B12 is a primary response gene in HUVE cells as evidenced by its rapid induction (within 1 h) which occurs in the absence of intervening protein synthesis. TNF treatment in the presence of CHX further increases the level of B12 transcript approximately 2-fold. The induction of B12 by TNF itself is mediated at least in part by transcriptional activation of the gene as determined by nuclear run-on (Fig. 1C).

B12 gene expression is also regulated by mediators of in- flammation other than TNF (IL-lP and LPS), and the message is induced to about the same level as by TNF itself. This is also observed with the induction of other TNF-induced genes (26) and reinforces the similarity in the effects elicited by IL-lP, LPS, and TNF. This is not altogether surprising since LPS can activate monocytes to produce TNF (1) and also can induce IL-lP biosynthesis in HUVE cells (57). In- triguingly, the temporal and organ-specific expression of B12 during development raises the distinct possibility that TNF or other proinflammatory cytokines may play an important role in embryogenesis by influencing the expression of primary response genes that have been thought to be restricted to an inflammatory response. Considering that both inflam- mation and embryogenesis share numerous common features, including cell migration and tissue remodeling, it is likely that factors which regulate one process will also play a significant role in the other.

The lack of significant hydrophobic amino acid stretches and the likely intracellular localization of B12 effectively limit the potential functions to a participation in intracellular events. Although the data presented here do not implicate


function in an obvious manner, several lines of evidence suggest more than a housekeeping role for B12. The likelihood that B12 serves a regulatory role is supported by three find- ings. First, the rate at which the B12 protein is degraded parallels that of other regulatory proteins, particularly the oncogenes c-myc, ElA, v-myb, and c-fos, the tumor supressor p53, and the heat shock protein HSP70 (58). All of these proteins are involved either in oncogenic transformation (59) or the stress response (60). Interestingly, the protein sequence of these genes and B12 share the PEST sequence which appears to correlate with rapid degradation of the protein.

Second, the existence of a cluster of charged residues has been found to correlate with proteins involved in regulatory functions including transcription and replication factors, steriod and thyroid hormone receptors, heat shock proteins, nuclear proto-oncogenes, and transforming proteins (61). The charge cluster situated in the 36 carboxyl-terminal amino acids of B12 contains 11 basic and 9 acidic residues, conferring a net positive charge. This grouping of charges differs from those found in transcriptional factors in that the clusters in these proteins have at least a 2:l basic/acidic charged residue ratio and that the cluster is usually located NH2-terminal to a DNA-binding domain. Charge clusters in steriod and thyroid hormone receptors invariably occur in the central portion of the molecule, within or carboxyl-terminal to the DNA-binding finger region. Thus the B12 charge cluster differs from those found in these classes of DNA-binding proteins. The charge clusters found in heat shock proteins are variable in location and net charge, but generally have a higher charge density/ cluster than the 58% charge content seen in the carboxyl terminus of B12 (62). Therefore, although it is possible that the charge cluster found in B12 has a regulatory function, it is unique in that it is not easily classified into any of the catagories mentioned above.

Third, proteins with amphipathic helices most often either interact with membrane phospholipids (50) or are DNA- binding proteins of the helix-loop-helix class (63). It is unlikely that B12 is a member of the latter class of proteins since the B12 sequence predicts the existence of only a single amphipathic helix, although it is possible that B12 may be able to interact with these DNA-binding proteins through this single amphipatic helix in some regulatory role.

Subcellular localization of B12 by immunostaining in unstimulated and TNF-stimulated HUVE cells will help narrow the list of possible functions for this protein. Initial attempts at immunolocalization by immunoperoxidase staining and immunofluorescence with the polyclonal B12 antisera have been unsuccessful, possibly either due to the small amount of B12 antigen present, or due to the quality of the antibody itself. It may be necessary to raise antisera to another region of the molecule for successful immunolocalization.

In summary, we have described the cloning and characterization of a novel TNF-inducible primary response gene which encodes an expressed 36-kDa protein. Extensive database searches established that this gene encodes an entirely novel protein product which appears to contain structural features which are shared by other cellular regulatory proteins. That the B12 gene is highly conserved through evolution (Fig. 5) and encodes a PEST protein with a short half-life suggests that B12 plays an essential regulatory role in the cell.

Acknowledgments-We thank Dr. Mark Boguski for assisting with the computer searches, and Karen O’Rourke, Larry Holzman, and Roger Eddy for assistance and discussions.

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