Proc. Natl. Acad. Sci. USA
Vol. 96, pp. 4518–4523, April 1999
A novel lipopolysaccharide-induced transcription factor regulating
tumor necrosis factor ? gene expression: Molecular cloning,
sequencing, characterization, and chromosomal assignment
FUMIO MYOKAI*, SHOGO TAKASHIBA*, ROGER LEBO†, AND SALOMON AMAR*‡
*Boston University, Department of Periodontology and Oral Biology, School of Dental Medicine, Boston, MA 02118; and†Center for Human Genetics, Boston,
Communicated by Susan E. Leeman, Boston University School of Medicine, Boston, MA, February 18, 1999 (received for review December 16, 1998)
tor of monocytes and macrophages, causing secretion of tumor
necrosis factor ? (TNF-?) and other inflammatory mediators.
Given the deleterious effects to the host of TNF-?, it has been
The nature of the nuclear factor(s) that control TNF-? gene
transcription in humans remains obscure, although NF-?B has
been suggested. Our previous studies pertaining to macrophage
response to LPS identified a novel DNA-binding domain located
transcriptional activity, but lacks any known NF-?B-binding
sites. We have used this DNA fragment to isolate and purify a
60-kDa protein binding to this fragment and obtained its amino-
terminal sequence, which was used to design degenerate probes
to screen a cDNA library from THP-1 cells. A novel cDNA clone
(1.8 kb) was isolated and fully sequenced. Characterization of
this cDNA clone revealed that its induction was dependent on
LPS activation of THP-1 cells; hence, the name LPS-induced
TNF-alpha factor (LITAF). Inhibition of LITAF mRNA expres-
sion in THP-1 cells resulted in a reduction of TNF-? transcripts.
In addition, high level of expression of LITAF mRNA was
observed predominantly in the placenta, peripheral blood leu-
kocytes, lymph nodes, and the spleen. Finally, chromosomal
LITAF mapped to chromosome 16p12–16p13.3. Together, these
findings suggest that LITAF plays an important role in the
activation of the human TNF-? gene and proposes a new
mechanism to control TNF-? gene expression.
Lipopolysaccharide (LPS) is a potent stimula-
The innate host response to bacterial pathogens is characterized
by an immediate release of biologically active compounds, in-
cluding monokines and cytokines. These proinflammatory mol-
ecules, which are intended to enable the host to eliminate the
pathogen, may adversely affect the host. Endotoxins, produced
from the outer membrane of Gram-negative bacteria, and exo-
toxins, released from the cell wall of Gram-positive bacteria, are
Lipopolysaccharide (LPS), extracted from the outer membrane
of Gram-negative bacteria, has been identified as a principal
secretion of nitrogen intermediates, prostaglandins, and cyto-
kines. Secretion of tumor necrosis factor ? (TNF-?), IL-1, IL-6,
and IL-12, demonstrated both in vivo and in vitro (3, 4), leads to
(5–8). In turn, other cells are induced to produce large quantities
(6, 7, 9), synthesis of acute-phase proteins (10), release of
collagenase (8), and synthesis of prostaglandin (11). In acute
situations, the pathogen often is eliminated, with resolution of
inflammation and minimal tissue damage. However, failure to
control the pathogen often leads to a state of metabolic anarchy
in which the inflammatory response is not controlled and signif-
icant tissue damage results.
Although the inflammatory response is mediated by a variety
of secreted factors, the cytotoxic effects of LPS have been
ascribed to TNF-? activity (4, 12, 13). TNF-? is a pleiotropic
cytokine and may benefit the host or exert detrimental effects on
the host (14–16). TNF-? helps prevent cancer (17), protects
against infection (18–21), promotes tissue remodeling (22), and
activates inflammatory responses (23). Conversely, TNF-? me-
diates septic shock in chronic infections (4, 24, 25), is responsible
for cachexia in cancer patients (26), causes inflammation in
The pleiotropic effects of TNF-? are dose-dependent (33).
Hence, the perceived need to control TNF-? production has
raised interest into the understanding of the mechanisms that
modulate TNF-? gene expression.
It is well known that gene transcription is controlled by
DNA-binding proteins (34). Recently, several groups have ex-
amined the transcriptional regulation of TNF-? by various in-
ducers, such as virus, LPS, and phorbol 12-myristate 13-acetate
(PMA) (35–45). The human TNF-? promoter contains motifs
to the involvement of NF-?B in TNF-? gene regulation. These
nor do they appear to be able to stimulate virus or LPS induction
alone (35). However, it has been suggested that NF-?B is an
important factor in TNF-? gene transcription in LPS-challenged
monocytes and macrophages. NF-?B-binding motifs are found in
the human TNF-? promoter region (46, 47) and were shown to
translocate into the nuclei of LPS-stimulated monocytes (36, 48,
49). In mice, mutation(s) or deletion(s) of NF-?B-binding motifs
on the TNF-? promoter failed to show reporter gene activation
in transfected cells (37, 50, 51). However, in humans, TNF-?
promoter activity in transfected cell lines was found to be
independent of the NF-?B-binding motifs (52). Drouet et al. (37)
offered an explanation for these conflicting data, suggesting that
enough NF-?B is constitutively expressed to sustain high-level
baseline expression of the human TNF-? gene (52) compared
with the mouse. Nonetheless, the nature of the nuclear factor(s)
in humans remains unknown.
Our previous studies pertaining to macrophage response to
LPS have identified a DNA sequence domain located from ?550
to ?487 in the human TNF-? promoter (53). Using electro-
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PNAS is available online at www.pnas.org.
Abbreviations: TNF, tumor necrosis factor; PMA, phorbol 12-
myristate 13-acetate; LPS, lipopolysaccharide; LITAF, LPS-induced
TNF-alpha factor; FISH, fluorescence in situ hybridization; UTR,
Data deposition: The sequence reported in this paper has been
deposited in the GenBank database (accession no. U77396).
‡To whom reprint requests should be addressed at: Boston University,
100 East Newton, G05, Boston, MA 02118. e-mail: firstname.lastname@example.org.
phoretic mobility-shift assays and selective mutations, it was
shown that a 64-bp fragment located within this region can bind
unknown protein(s) and this binding is responsible for TNF-?
transcriptional activity. Sequence analysis of this fragment re-
vealed the absence of any potential NF-?B-binding sites (53).
These intriguing results suggested the existence of a novel cis-
acting regulatory protein, other than NF-?B, that is necessary for
human TNF-? gene transcription.
In an effort to further our understanding of the molecular
mechanisms of LPS-induced TNF-? gene regulation, we used
this 64-bp fragment to isolate the purported native protein. Its
amino-terminal sequence was obtained and used to design
PCR primers to screen a THP-1 cDNA library. Using these
probes, we isolated a novel full-length cDNA (1.8 kb) with a
deduced amino acid sequence of the ORF encoding 229 aa.
Characterization of this new gene revealed that its expression
resulted from LPS stimulation of THP-1 cells; hence, the name
LPS-induced TNF-alpha factor (LITAF). We investigated
tissue distribution of the LITAF gene and found transcripts in
a variety of human adult tissues. Northern blot hybridization
revealed a high level of expression in the placenta, peripheral
blood leukocytes, lymph nodes, and the spleen. Inhibition of
LITAF mRNA translation using antisense DNA in naive
THP-1 cells or those exposed to LPS resulted in a reduction of
TNF-? transcripts. In addition, chromosomal localization us-
ing fluorescence in situ hybridization (FISH) revealed that
LITAF mapped to chromosome 16p12–16p13.3. Together,
these findings suggest that the LITAF gene may play an
important role in human TNF-? gene activation.
MATERIALS AND METHODS
Cell Culture. The human monocytic cell line, THP-1, was
maintained in complete RPMI [C-RPMI; RPMI 1640 medium
supplemented with 2 mM L-glutamine?25 mM Hepes?100
units/ml of penicillin?100 mg/ml of streptomycin?10% FBS (all
Preparation of Nuclear Extracts from Cultured THP-1 Cells.
THP-1 cells were induced to maturation by incubation in 200 nM
PMA (Sigma) for 20 hr and then stimulated with 100 ng?ml of
Porphyromonas gingivalis LPS (54) for 2 hr. Nuclear extracts were
prepared as described in our previous work (53).
Preparation of DNA Affinity Beads. The 64-bp initiator ele-
ment located from –550 to ?487 in the human TNF-? promoter
(55) was amplified by PCR. The PCR mixture (50 ml) was
prepared as described previously (55). The human TNF-? pro-
moter (55) was used as the template for the PCR. We used the
sequence 5?-TGAGGCCTCAAGCTGCCACCA-3? for the for-
ward primer sequence and 5?-XTGAGGCCTGTGTTT-
GGGTCTG-3? for the reverse sequence (X ? 5? biotin). The
cycling parameters (30 cycles) were as follows: an initial dena-
turation at 94°C for 1 min, annealing at 55°C for 1 min, and
elongation at 72°C for 1 min. The last cycle had an additional
elongation time of 7 min. The PCR products were separated on
unincorporated biotinylated primers were removed by using the
QIAEX II Gel Extraction Kit (Qiagen). The biotinylated DNA
was desalted and dissolved in Tris?HCl?EDTA (2 mg?ml) and
then used for the preparation of DNA affinity beads. Immobili-
zation of labeled DNA to Dynabeads M-280 streptavidin (10
mg?ml; Dynal, Great Neck, NY) was performed as described by
Gabrielsen and Huet (56). Briefly, a suspension of beads was
coupled with the labeled DNA at room temperature for 30 min.
The affinity beads were washed to remove unbound DNA and
then used for purification of DNA-binding protein.
Purification of DNA-Binding Protein Using Affinity Beads.
described previously (56). Briefly, the nuclear extracts were
incubated with the beads in binding buffer containing poly
(dI-dC) at room temperature. After washing (twice), the DNA-
binding protein was eluted, desalted, and then used for further
Isolation of the DNA-Binding Protein and Sequence Analysis.
The eluted protein sample was separated on 12% SDS-
polyacrylamide gel as described previously (53). The resolved
proteins (approximately 2% of the protein of the total protein)
were electroblotted onto a polyvinylidene difluoride membrane
a 0–100% acetonitrile gradient containing 0.01% trifluoroacetic
acid (58). Fractions were collected, and a number of peptides
were sequenced by established methods as described previously
(57). All peptide sequences were found in the isolated cDNA
clone. Degenerate oligonucleotides (16-mer) corresponding to
either end of peptide 1, M-S-V-P-G-P-Y-Q-A-A-T-G, were used
for PCR by using the LPS-induced THP-1 cDNA as a template
to obtain a DNA fragment containing the exact nucleotide
sequences encoding the middle part of this peptide. PCR prod-
ucts resolved in an 8% acrylamide gel were eluted by using the
QIAEX II Gel Extraction Kit (Qiagen) and subcloned into a
Screening the cDNA Library. An LPS-stimulated, PMA-
induced, custom-made human THP-1 Uni-ZAP XR cDNA
library (Stratagene) was screened by using this PCR probe, and
nine positive clones containing 0.8- to 1.8-kb inserts were ob-
tained after screening 5 ? 105colonies. The longest cDNA insert
(1.8 kb) was chosen for complete sequencing after the
dideoxynucleotide chain-termination method by using a 373A
sequencer (Applied Biosystems).
The BLAST program was used to search the database through
the National Center for Biotechnology Information. The PILEUP
and PRETTYBOX programs were used for sequence alignments
and comparisons. The FASTA program was used for calculating
Wisconsin Genetics Computer Group, Madison, WI. Finally, the
SIGSCAN program was used to search the Transcription Factor
Database (TFD) through the BIMAS program of the Advanced
Biosciences Computing Systems maintained at the University of
Detection of LITAF Gene Transcripts. The transcripts for the
new gene were detected by Northern blot analysis in cultured
were induced to maturation by incubation in 200 nM PMA
(Sigma) for 20 hr and then stimulated with 100 ng?ml of P.
gingivalis LPS (54) for 2 hr. Noninduced THP-1 cells also were
cultured. Total cellular RNA was collected by using RNA
STAT-60 (Tel-Test, Friendswood, TX) by the method described
in the instruction manual, and poly(A)?RNA (mRNA) was
and transferred onto a Hybond-N?membrane (Amersham) as
described previously (59). Northern blot filters with mRNA from
several different human tissues were obtained from CLON-
TECH. These filters were hybridized with an35S-labeled cRNA
probe at 60°C overnight in 50% formamide?5? SSC?5? Den-
hardt’s solution?0.1% SDS?50 mM sodium phosphate, pH 6.9?
heat-denatured salmon sperm DNA. The filters were washed
three times in 0.1? SSC?0.1% SDS at 68°C for 30 min and then
autoradiographed with BIOMAX MR film (Kodak). The 511 bp
of the HincII-ApaI cDNA fragment from the coding sequence in
the new gene (Fig. 1) was subcloned into pGEM7Zf(?) (Pro-
mega). The antisense and sense riboprobes were prepared from
this plasmid and labeled with [35S]UTP as described previously
(59). These probes were used for Northern hybridizations.
(pRc?CMV; Invitrogen) were constructed as described previ-
Immunology: Myokai et al.Proc. Natl. Acad. Sci. USA 96 (1999)4519
antisense or a sense orientation downstream of the human
cytomegalovirus promoter element as the first ORF. The vector
contained a neo-resistance gene for selection of stable transfec-
tants. Plasmid constructions were as follows: AS5?, antisense
cDNA corresponding to a 320-bp fragment that covered the 5?
untranslated region (UTR) and the AUG start codon; SE, sense
full-length of cDNA; and CON, control vector without insert.
These constructions are shown in Fig. 1. All plasmids were
prepared by using Qiagen Plasmid Midiprep Kits.
Stable Transfection. THP-1 cells (5 ? 106?cuvette) were
transfected by electroporation (Gene Pulser; Bio-Rad) with 20
mg of recombinant plasmid according to the manufacturer’s
instructions. Mock cells were transfected similarly with a control
containing 0.8 mg?ml of G418 (Geneticin; GIBCO). This me-
dium was changed every 3 days. The surviving cells were used for
transformation was confirmed by Northern blot analysis with
antisense and sense riboprobes. Total cellular RNA was recov-
ered from transfected cells (2 ? 106) and used for Northern blot
analysis in a manner similar to that described previously. The 707
and a coding sequence for the new gene (Fig. 1), was subcloned
into pBluescript SKII(?). The35S-labeled riboprobes for hybrid-
ization were prepared from this plasmid as described previously
Quantification of TNF-? mRNA Expression in Transfected
Cells. TNF-? mRNA expression was quantified by using an
incubated with 200 nM of PMA (Sigma) for 20 hr to induce
LPS (54) for 2 hr. mRNA was recovered from the cells as
described previously. Successful recovery of mRNA from each
sample was monitored by Northern blot hybridization with a
?-actin probe. The 355-bp TNF-? cDNA was obtained from a
agene) by PCR with specific primers for TNF-? mRNA (Strat-
agene). After the cDNA was subcloned to a SmaI site of
pGEM7Zf(?), the TNF-? cDNA was identified by sequencing.
After the plasmid was linearized with XbaI and transcribed with
SP6 polymerase, a 446-bp35S-labeled antisense probe was con-
structed. The RNase protection assays were performed as de-
scribed previously (59).
with biotin by using kit no. S4099 (Oncor) according to the
DNA and 75 ?g of salmon sperm DNA were resuspended in 50
?l of hybrisol VII (catalog no. S1390-10; Oncor) by sonication.
for 30 min for biotin labeling before mixing with the D16Z1
Probe (catalog no. P5035-DG-5; Oncor) was denatured at 70°C
for 5 min and used to confirm the location of the LITAF gene on
chromosome 16. Normal lymphocytes were treated with colce-
according to standard cytogenetic protocols. The slides were
denatured in 2? SSC (pH 7.0) plus 70% formamide at 72°C for
volumes of labeled, denatured probes were mixed, and 20 ?l was
added to each dehydrated slide. Hybridization proceeded for
15–17 hr at 37°C. The slides were washed for 15 min at 43°C in
2? SSC plus 65% formamide and then at 37°C for 8 min in 2?
SSC. The biotin-labeled probe was detected by binding to avidin-
Texas red, washed, amplified with antiavidin, washed, and am-
plified with avidin-Texas red by using the Biotin-Texas Red
Detection Kit (catalog no. S1334-BTR; Oncor) according to the
manufacturer’s instructions. The digoxygenin-labeled hybridized
probe was detected with FITC-labeled antidigoxygenin antibody,
washed, amplified with rabbit-anti-sheep antibody, washed, and
amplified with FITC-anti-rabbit antibody (Digoxygenin-FITC
Detection Kit, Cat. No. S1331-DF) according to the manufac-
turer’s instructions. The slides were counterstained with 4?,6-
diamidino-2-phenylindole and photographed by using triple-
excitation?emission bandpass filters (Cat. No 61002X; Olympus,
New Hyde Park, NY).
Screening, Sequencing, and Structural Analysis of LITAF
cDNA. As shown in Fig. 2, the cDNA consists of a 234-nt 5?
noncoding region, a 687-nt ORF, and an 852-nt 3? noncoding
23.9-kDa protein (GenBank accession no. U77396). At the time
that the DNA sequencing was almost completed, the clone was
entered in GenBank as TNF-?-induced mRNA and did not have
any homology with nucleotide and protein sequences available in
all public databases. Polyak et al. (62) then examined transcripts
markedly induced by the p53 gene. Among them, an expressed
sequence tag named PIG7 was found to harbor 98% homology
renamed this cDNA LITAF because it is induced by LPS and
affects TNF-? gene expression.
Induction of LITAF Gene mRNA. Northern blot analysis
clearly indicated that a single 1.8-kb mRNA encoding the LITAF
gene was present in LPS-induced PMA-differentiated THP-1
cells. The size of the transcript was consistent with the sequence
data. The expression of the LITAF gene occurred only in
was found in PMA-differentiated cells in the absence of LPS
stimulation, in LPS-stimulated cells in the absence of PMA
differentiation, or in unstimulated THP-1 cells (Fig. 3).
Expression Patterns of the Novel Gene in Human Tissues. As
shown in Fig. 4, a 1.8-kb transcript was significantly expressed in
spleen, lymph node, and peripheral blood leukocytes. Moderate
expression was observed in thymus, appendix, bone marrow,
kidney, and placenta. Little expression was found in pancreas,
skeletal muscle, liver, and lung whereas a very minor expression
was observed in heart, brain, and fetal liver. The size of the
transcripts again was consistent with the sequence results and
confirms that the cDNA isolated was the full-length clone.
Antisense and Sense LITAF mRNA Constructs and Expres-
sion. To determine the biological activity of the isolated LITAF
gene, THP-1 cells were transfected with the following constructs:
LITAF antisense RNA (AS5?), LITAF sense RNA construct(s),
transcript was detected in sense-transfected cells (Fig. 5A), and
cDNA isolated. The dark, thicker line denotes the ORF (from AUG 234
to UAG 918) in LITAF. Restriction sites for EcoRI, HincII, PstI, SacI,
and ApaI are shown. Note that the antisense RNA represented as AS5?
was designed to be complementary to the 5? region of LITAF mRNA
LITAF was used as sense RNA for overexpression of LITAF RNA and
is shown as SE. (B) Schematic structure of antisense or sense constructs
inserted downstream of the cytomegalovirus promoter.
cDNA cloning of LITAF. (A) Physical map of the LITAF
4520Immunology: Myokai et al.Proc. Natl. Acad. Sci. USA 96 (1999)
using LITAF sense RNA as a probe, we detected a band of
approximately 400 bp in antisense-transfected cells (Fig. 5B). No
signal was detected in mock-transfected cells (Fig. 5). These
results are consistent with the antisense construction design.
Subsequent examination of the effect of inhibiting LITAF
RNA using LITAF antisense constructs on the transcription of
the human TNF-? gene revealed that TNF-? mRNA signals
were reduced more strongly in LITAF antisense-expressing
cells than in mock-transfected cells (Fig. 6). No changes in
TNF-? mRNA signals were observed with LITAF sense-
expressing cells compared with mock-transfected cells (Fig. 6).
Similar amounts of ?-actin mRNA were found in LITAF
antisense-expressing cells, LITAF sense-expressing cells, and
mock-transfected cells (data not shown).
Chromosome Localization. FISH of the LITAF cDNA probe
to human metaphase spreads resulted in specific labeling on
chromosome 16 (Fig. 7). The location on chromosome 16 ?-sat-
D16Z2 (Oncor). The probe had specificity for this site, because
symmetrical signals were not observed on other chromosomes.
The FISH signals were localized relative to the chromosome
bands and the LITAF gene locus assigned to chromosome 16
The present paper reports the cloning, characterization, and
that affects TNF-? gene expression in cells of monocytic lineage.
The cDNA encoding LITAF was isolated by screening an LPS-
induced THP-1 expression library with degenerate oligonucleo-
tides corresponding to the native nuclear protein isolated. Func-
by using antisense RNA in PMA-differentiated THP-1 cells
exposed to LPS resulted in a reduction of TNF-? transcripts. In
humans this gene has been localized to chromosome 16p12–
All the public nucleotide and protein sequence databases were
searched. None of the sequences in the coding region were found
to be similar to typical DNA-binding motifs. We did find se-
mRNA from THP-1 cells. The cells (2 ? 107)
were cultured in various conditions: stimula-
tion with P. gingivalis LPS (LPS); differentia-
tion with PMA (PMA); differentiation with
PMA followed by stimulation with LPS
mRNA was recovered from cells, run on
denaturing formaldehyde?1.2% agarose gel,
and transferred to a Hybond-N?filter. The
filter was hybridized with the antisense RNA
probe that corresponded to the coding region
of the LITAF gene, as described in Materials
and Methods. The hybridized filter was ex-
posed to x-ray film for 24 hr. A 1.8-kb transcript was observed only in
PMA?LPS-stimulated THP-1 cells. Similar amounts of ?-actin mRNA
were found in all mRNA tested (data not shown).
Northern blot hybridization of
fected with AS5?, SE, or control vector (MOCK). Total RNA was
recovered from 2 ? 106cells after differentiation with PMA and
stimulation with P. gingivalis LPS. (A) RNAs were hybridized with the
LITAF sense RNA probe. (B) RNAs were hybridized with the LITAF
antisense RNA probe. Similar amounts of ?-actin mRNA were found in
LITAF antisense-expressing cells, LITAF sense-expressing cells, and
mock-transfected cells (data not shown).
Northern blot hybridization of RNA from THP-1 cells trans-
sequence. The full-length LITAF cDNA was isolated from a cDNA
library of PMA-differentiated THP-1 cells stimulated with LPS. This
sequence has been submitted to GenBank and has been assigned acces-
sion no. U77396. The ORF encodes 228 aa with a predicted molecular
mass of 23.9 kDa. The Alu sequence is underlined.
Nucleotide sequence of LITAF and its deduced amino acid
adult tissues. (A and B) Expression of the LITAF gene. The preblotted
corresponded to the coding region of the LITAF gene, as described in
Materials and Methods.
Northern blot hybridization of mRNA (2 ?g) from different
Immunology: Myokai et al.Proc. Natl. Acad. Sci. USA 96 (1999) 4521
quences in two other regions of the gene (5? and 3? UTRs) that
displayed some homology with sequences reported in the data-
bases: several amino acid sequences were found to be similar to
the Bicaudal-C gene of Drosophila melanogaster. Although the
Bicaudal-C gene product contains a motif called the KH domain,
which is found in many RNA and single-stranded DNA-binding
proteins (63), we did not find a typical KH domain in the LITAF
gene. In addition, sequencing of the LITAF gene revealed Alu
have reported that Alu elements constitute approximately 5% of
the human genome and often are found in introns or 3? UTRs.
However, the Alu region mistakenly may be incorporated into
ORFs (84). To determine whether the presence of the Alu
element was a result of alternative splicing from an adjacent
intron, the other two cognate clones were sequenced. We found
that the nucleotide sequences of the cognate clones significantly
overlapped, indicating that the presence of the Alu region in the
cDNA probably was not caused by a splicing error.
Recently, Neuenchwander et al. (64) reported that stable
antisense RNA expression inhibits the translation of sense RNA.
In a number of studies (65–68), stable antisense RNA expression
the mechanism responsible for this decrease remains unclear, it
may be related to the inhibition of RNA synthesis, RNA splicing,
subunits, and sliding of the ribosome along the mRNA coding
sequence, all of which result in translation arrest (69). Therefore,
level by transfecting antisense oligonucleotide-producing vectors
into THP-1 cells. The most common mRNA target site reported
in the literature is the AUG translation initiation codon (70).
However, we have found in many cases that oligonucleotides that
target other regions in the mRNA, such as 5? and 3? UTRs, were
more effective (71–74). Therefore, to interfere with LITAF
mRNA, we transfected vectors producing antisense oligonucle-
overexpression of the LITAF mRNA, a vector producing the
full-length LITAF mRNA was transfected. Using sense LITAF
RNA as a probe, a band of approximately 400 bp was detected in
AS5?-transfected cells, consistent with the design of the antisense
vector. No significant signal was detected in mock-transfected
cells. When the full-length sense LITAF mRNA was transfected
in THP-1 cells, an intense signal for LITAF mRNA was detected
at 1.8 kb by using an antisense LITAF mRNA probe, reflecting
expression of RNA from each construct. However, when the
expression with the antisense was not complete and LITAF
the LITAF gene partly as a compensatory mechanism to restore
the function, although the mechanisms involved in this phenom-
ena remain unknown.
Using the RNase protection assay, examination of the effect of
antisense RNA on transcription of the human TNF-? gene
revealed that the signal indicating the TNF-? mRNA level was
weaker in antisense-expressing cells than in mock cells. However,
similar amounts of ?-actin mRNA were found in both cells. The
reduction of TNF-? mRNA that occurs when LITAF gene
mRNA activity is arrested strongly suggests that LITAF gene
products play an important role in the regulation of human
TNF-? gene transcription. In addition, the expression level of
TNF-? mRNA was not enhanced in the sense-expressing cells
(Fig. 6). The scope of our functional study is not broad enough
for us to rule out the possibility that exogenous LITAF gene
products may regulate TNF-? gene transcription in other ways
than what occurs naturally. We posit three possibilities for how
the LITAF gene product may regulate gene transcription: (i) the
TNF-? gene; (ii) the endogenous LITAF gene product is enough
to activate the TNF-? gene; and (iii) the exogenous LITAF gene
product acts after protein processing because a large amount of
LITAF mRNA is constitutively transcribed in the cells (Fig. 5).
LPS induction of TNF-? promoter may be mediated by the
concerted participation of at least two separate, cis-acting regu-
latory elements (75).
Distribution of LITAF mRNA on various tissue blots revealed
that the LITAF gene is expressed in most of the tissues tested;
however, it is expressed more predominantly in hematolympho-
poeitic tissues and placenta, kidney, and pancreas. This distribu-
tion of LITAF transcripts seems to parallel TNF-? tissue distri-
bution during endotoxemia (76, 77).
The potential role of LITAF in human disease is implicated by
the recent finding that p53 induces the expression of the LITAF
gene (62) and by its chromosomal localization at 16p12–16p13.3.
The p53 tumor-suppressor protein is thought to play a major role
in the defense of the cell against agents that damage DNA (78,
79). Loss of function of the p53 tumor-suppressor gene is a
frequent and important event in the genesis or progression of
many human malignancies. Loss of p53-dependent apoptosis is
believed to be critical to carcinogenesis in many of these cases,
suggesting the possibility to therapeutically restore this pathway
and directly eliminate malignant cells or increase or restore their
sensitivity to chemotherapeutic agents (78, 79). The regulation of
p53-dependent responses is complex and variable between cell
types, and whether a cell undergoes apoptosis after activation of
p53 is highly sensitive to signal context, including environmental
and cell-intrinsic influences. Further insight has been provided
into the activation of latent p53, the biochemical mechanisms
involved in growth arrest and apoptosis, and the influence of
RNA from THP-1 cells transfected with
AS5?, SE, or control vector (MOCK).
Total RNA was recovered from 2 ? 106
cells after differentiation with PMA and
stimulation with P. gingivalis LPS. The
RNAs were hybridized with the TNF-?
antisense RNA probe. After treatment
with RNase A and T1, protected bands
were electrophoresed through an 8%
polyacrylamide gel, and the dried gel was
exposed to x-ray film. No protected band
is observed in AS5?-transfected cells. The
band density observed in SE-transfected
MOCK-transfected cells. Left lane, native
TNF-? antisense probe.
RNase protection assay of
banding is observed on chromosome 16p12–16p13.3.
FISH localization of human LITAF gene. Metaphase chro-
4522Immunology: Myokai et al.Proc. Natl. Acad. Sci. USA 96 (1999)
and processes that may contribute directly to the maintenance of
genomic stability (78, 79). Recently, various monocytic cell lines
have been shown to respond to LPS and interferon ? with
endogenous nitric oxide (NO) formation (80), which, in turn,
activates the expression of p53, leading to apoptosis (81). Fur-
thermore, p53 has been shown recently to modulate the activity
of various inflammatory cytokines such as IL-6 (82), IL-2, and
IL-4 (83). In light of the present data, a potential, new p53-
dependent pathway affecting TNF-? gene expression can be
NO production. In turn, NO can up-regulate p53, which would
stimulate the expression of the LITAF gene. Ultimately, the
induction of the LITAF gene product would affect TNF-? gene
expression. The validity of this pathway remains to be substan-
product might be a crucial factor for the control of TNF-? gene
This work was supported by National Institute of Craniofacial and
Dental Research Grant DE10709 to S.A.
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