From the
A major part of the anti-inflammatory effect of glucocorticoids
is attributable to their attenuation of the induction of genes whose
products mediate intercellular interactions, e.g. cytokines
and the inducible forms of prostaglandin synthase and nitric oxide
synthase. We hypothesized that (i) there exists a class of
immediate-early/primary response genes whose induction by inflammatory
agents, mitogens, and other stimuli is attenuated by glucocorticoids,
and (ii) the products of these glucocorticoid-attenuated response genes
(GARGs) function predominantly in paracrine cell processes. We
constructed a
Of the
five GARG cDNAs not previously known, one encodes a novel member of the
CXC chemokine family, designated LIX (LPS-induced CXC
chemokine). The predicted LIX protein has a 40-amino acid signal
sequence and a 92-amino acid mature peptide with a distinctive
COOH-terminal region. Surprisingly, segments of the 3`-untranslated
regions of LIX and two other CXC chemokines have substantially
greater nucleotide sequence homology than do their coding regions.
These segments may perform an unknown regulatory function. The LIX
message is strongly induced by LPS in fibroblasts, but not in
macrophages, suggesting that LIX may participate in the recruitment of
inflammatory cells by injured or infected tissue.
The message levels of primary response/immediate early genes
increase in response to cellular stimulation, even in the presence of a
protein synthesis inhibitor(1) . Transcription of primary
response genes is induced through signal transduction cascades that
activate latent, pre-existing transcription factors. Many primary
response genes (e.g.jun, fos, myc, egr-1/TIS8) encode transcription factors that function
intracellularly in growth control and cellular differentiation. Others
encode proteins involved in paracrine intercellular communication.
These include the inducible prostaglandin synthase (TIS10/PGS2)
Induction of
primary response genes encoding transcription factors involved in the
G
To
test our proposals, we screened 15,000 clones of a cDNA library by
differential hybridization, using lipopolysaccharide (LPS) as the
inducer and dexamethasone (DEX) as the glucocorticoid. The
characteristics of the 12 distinct GARG cDNAs we identified support
both hypotheses: five GARG cDNAs are previously undescribed sequences;
and of the seven previously known sequences, six encode secreted
products with known functions in intercellular communication. One of
the five novel GARG cDNAs we cloned encodes a new member of the
CXC chemokine family, which we designated LIX, for LPS-induced
CXC chemokine.
Mice used in experiments reported in this
paper were maintained and handled in accordance with institutional
guidelines. C57B/6 mice (6-8 weeks old) were given
intraperitoneal injections with LPS or sterile saline and 4 h later
were anesthetized with ether and killed by cervical dislocation.
Phage from the cDNA
library were plated at a density of
The
four CXC chemokines most similar to LIX are more closely
related to each other than to LIX. These peptides, porcine alveolar
macrophage chemotactic factor-II (AMCFII)(47) , human and bovine
granulocyte chemotactic peptide-2 (GCP2)(48) , and human
epithelial neutrophil activating peptide-78 (ENA-78)(49) , have
only 47-54% amino acid identity with LIX (), but
there is 78% identity between porcine AMCFII and bovine GCP2, 67%
identity between bovine GCP2 and human GCP2, and 74% identity between
human GCP2 and ENA-78. All four differ from LIX at 15 sites. At eight
of these sites, all four peptides are identical to one another. At the
remaining seven sites, two or more have an identical residue. The
Gln-51 residue in LIX is unique: all other known CXC peptides
have Gly at the corresponding position, except for Asp in crg2 ( Fig. 5and sequences not shown). These amino acid differences,
plus the distinctive COOH-terminal region, suggest that the LIX is a
novel CXC chemokine.
In
addition to glucocorticoid attenuation, our screening strategy involved
pretreatment with TGF-
Previous searches for primary
response genes induced by mitogens or inflammatory agents have yielded
a large proportion of transcription factors and other intracellular
proteins, as well as secreted proteins (1, 52). In contrast, our
screening yielded six secreted proteins among the seven known GARG
cDNAs we cloned. Furthermore, both of the inducible non-GARG cDNAs we
cloned encode products, a transcription factor and a cytoskeletal
protein, with intracellular functions. The very high proportion of
secreted proteins among the known GARGs we cloned is consistent with
our proposal that glucocorticoid attenuation defines a functional
subclass of primary response genes (inducible by LPS or by other
mediators) whose products are predominantly involved in extracellular
rather than intracellular processes. It should be noted, again, that
such gene products need not be secreted, as exemplified by TIS10/PGS2
and iNOS.
The function of LIX is unknown. Near
its predicted NH
Another
regulatory function mediated by 3`-untranslated sequences is message
destabilization, which often involves AU-rich sequences containing or
adjacent to the pentamer
AUUUA(57, 58, 59, 60) . The LIX mRNA
includes five AUUUA sequences, one of which is located within the
segment highly conserved among LIX, AMCFII, and ENA-78. This segment
might be the target of a specific AUUUA-binding protein.
In summary, the search for GARGs undertaken in this study
has led to the identification of five previously undescribed murine
cDNAs, including a new member of the CXC chemokine family. The
results also suggest the existence of many other
glucocorticoid-attenuated genes encoding intercellular mediators not
yet described. Continued identification and characterization of novel
GARG genes should be a fruitful approach for discovering other new
mediators of intercellular communication and for elucidating the
molecular mechanisms of glucocorticoid action.
This table summarizes the
results of data base searches using partial sequences of the GARG and
non-GARG cDNAs we cloned. Abundance is the number of cross-hybridizing
clones among the first 42 or 120 candidate phage. Insert size is
estimated from agarose gels with ethidium bromide staining. mRNA size
is estimated from northern blots and is consistent with the expected
values for the known genes. References for the identified genes are
given in the text.
The deduced
amino acid sequence of LIX was compared with the sequences of all
CXC chemokines found by searching gene and protein data bases
using the BLASTX program (18). Each sequence (excluding the signal
peptide) was aligned with LIX, and the percentage of identical residues
was calculated relative to the number of residues in the longer
peptide. The C-terminal length is the number of residues following the
fourth conserved cysteine.
The nucleotide sequence(s) reported in this paper has been
submitted to the GenBank
J. B. S. thanks Linda Vician, Rebecca Gilbert, and
other members of the Herschman laboratory for many useful discussions
and Tim Watanaskul for assistance with sequencing.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
cDNA library from transforming growth factor
1-pretreated murine Swiss 3T3 cells stimulated with
lipopolysaccharide (LPS) or serum in the presence of cycloheximide,
screened 15,000 plaques by differential hybridization, and cloned 12
LPS-induced, dexamethasone-attenuated cDNAs. Seven were previously
known. Six of these encode intercellular mediators (thrombospondin-1,
MCSF, JE/MCP-1, MARC/fic/MCP-3, crg2/IP-10, and cyr61); one encodes a
protein of unknown function (IRG2). Thus, a large majority of these
GARG cDNAs encode intercellular mediators, as hypothesized.
(
)and nitric oxide synthase (iNOS) enzymes and
cytokines such as JE/MCP-1 and KC/gro (1-3).
G
transition (e.g.fos, egr-1/TIS8, c-myc) is not
generally suppressed by glucocorticoids (4-7). In contrast,
glucocorticoids markedly attenuate TIS10/PGS2, iNOS, JE/MCP-1, and
KC/gro
induction(3, 4, 7, 8, 9, 10) .
These examples suggested to us that glucocorticoid attenuation may
distinguish a functional subclass of primary response genes involved in
intercellular communication. We hypothesized that TIS10/PGS2, iNOS,
JE/MCP-1, and KC/gro are representative of a larger class of primary
response genes whose induction by mitogens, growth factors, or
inflammatory stimuli is attenuated by glucocorticoids. We refer to such
genes as glucocorticoid-attenuated response genes, or GARGs. We also
hypothesized that GARGs predominantly encode proteins whose functions
are extracellular or intercellular, rather than intracellular.
Materials
Cycloheximide, dexamethasone, and
phenol-extracted lipopolysaccharide from Escherichia coliserotype 0111:B4 were purchased from Sigma; cell culture
media from Mediatech (Washington, D. C.); fetal bovine serum (FBS) from
Gemini Bioproducts (Calabasas, CA); restriction enzymes from New
England Biolabs (Beverly, MA); custom oligonucleotides from Integrated
DNA Technologies (Coralville, IA); and
[-
P]dCTP from DuPont NEN. Recombinant
TGF-
1 was a gift of A. Purchio (Bristol Myers/Squibb, Seattle,
WA).
Cell Culture and Animals
Murine Swiss 3T3
fibroblasts were cultured at 37 °C in 10% CO, air in
Dulbecco's modified essential medium supplemented with 10% FBS
and antibiotics. Near-confluent cultures were switched to medium
containing 0.5% FBS for 18-24 h before treatment with
dexamethasone, TGF-
1, LPS, or serum. These agents were added
directly to the culture medium. Mouse embryo fibroblasts were isolated
from strain 129 embryos and cultured in Dulbecco's modified
essential medium with 10% fetal bovine serum as described(11) .
RAW 264.7 macrophages were cultured in 5% CO
, air in RPMI
1640 medium with 10% FBS.
RNA Preparation and Northern
Analysis
Poly(A) RNA for library construction
and synthesis of cDNA probes was extracted directly from cell lysates
using biotinylated poly(dT) and streptavidin-coupled paramagnetic beads
(PolyATtract® kit, Promega, Madison, WI). Total cellular RNA for
Northern analysis was isolated with guanidinium
isothiocyanate(12) , subjected to electrophoresis on
formaldehyde-agarose gels, and transferred to charged nylon membranes
(Schleicher & Schuell) as described(13) . Prehybridizations
and hybridizations with
P-labeled probes were performed at
42 °C in 50% formamide, 0.65 M NaCl, 10
Denhardt's solution, 50 mM Tris, pH 7.5, 1% SDS, and 2
mM sodium pyrophosphate. Filters were washed at 65 °C in
0.5% SDS and 2
SSC (1
SSC is 0.15 M NaCl and
15 mM sodium citrate, pH 7.0) or in 0.1% SDS and 0.1
SSC. Filter autoradiograms were made by exposing XAR-5 film (Eastman
Kodak Co.) at -80 °C with one intensifying screen.
Radioactivity of specific bands was directly quantitated and analyzed
using a multiwire proportional counter system (Ambis Inc., San Diego,
CA). Corrections for small variations in loading of each lane were made
using radioactivity of the message for the constitutive ribosomal
protein S2 (CHOb).
Library Construction
For library preparation,
quiescent Swiss 3T3 cells were pretreated with TGF-1 (10 ng/ml)
for 1 h before addition of cycloheximide (10 µg/ml) and either FBS
(final concentration 20%) or LPS (10 ng/ml). Cells were harvested 2 and
4 h later. Two poly(A)
RNA preparations were utilized
in the synthesis of the library. One was purified from the combined 2
and 4 h lysates of LPS-treated cells, and the other from the combined 2
and 4 h lysates of serum-treated cells. These two poly(A)
RNA preparations were used separately as templates for
oligo(dT)-primed first strand cDNA synthesis, using the ZAP-cDNA
synthesis kit (Stratagene, La Jolla, CA). After second strand synthesis
and size fractionation to exclude cDNAs shorter than 500 bp, equal
quantities of the two cDNA preparations were combined, directionally
ligated into the
ZAP II vector, and packaged with Gigapack II
Gold (Stratagene). The primary library, which contained 2.9
10
plaques (95% recombinants), was amplified once to a
titer of 2.3
10
plaque-forming units/ml. Using
probes described in previous
studies(3, 13, 14) , we isolated phage
containing inserts for TIS10/PGS2, iNOS, S2, and egr-1/TIS8 to serve as
controls in the differential screening.
Differential Screening
Poly(A) RNA preparations used to prepare probes for differential
hybridization were obtained from three groups of Swiss 3T3 cells. Cells
for the ``plus'' probe were pretreated with 10 ng/ml
TGF-
1 for 1 h before addition of 10 ng/ml LPS. Cells for the
``minus'' probe were pretreated with 2 µM dexamethasone for 3 h before addition of 10 ng/ml LPS. Both groups
of cells were harvested after 2 h of continued incubation, along with
untreated cells (the third group).
P-Labeled cDNA probes
were synthesized with Moloney murine leukemia virus reverse
transcriptase (Promega) using both oligo(dT)
and
random hexamer primers (Pharmacia Biotech Inc.).
1900 clones on eight 150 mm
Petri dishes. Purified phage for S2, TIS10, and iNOS were spotted at
known locations on each plate to serve as controls. After overnight
growth of the phage, quadruplicate transfers to 0.45-µm
nitrocellulose filters (Schleicher & Schuell) were made from each
plate, with the filters applied sequentially for 1.5, 3, 6, and 12 min.
After air drying, the filters were vacuum-baked at 80 °C for
1-2 h and then preincubated in sealed plastic bags for 2-4
h at 42 °C in 50% formamide, 6
Denhardt's reagent, 6
SSC, 60 mM NaPO
(pH 6.8), 1 mM sodium pyrophosphate, 145 µg/ml rATP, and 60 µg/ml
sonicated nondenatured salmon sperm DNA. Equal counts of
P-labeled cDNA probes from the plus, minus, and uninduced
control cells were added to bags containing the first, second, and
third lifts, respectively, and hybridized at 42 °C for 4 days. The
fourth lift was hybridized overnight with
P-labeled probes
made by random priming from purified inserts for TIS10/PGS2 and iNOS
cDNAs. Filters were washed in 2
SSC + 0.5% SDS at room
temperature and then at 60 °C, with a final wash in 0.1
SSC
+ 0.1% SDS at 65 °C. Autoradiogram exposures of the plus,
minus, and control filters were adjusted when necessary to provide
equal signal intensity of the S2 control phage spots. Plaques were
chosen as candidates if they showed greater hybridization signal on the
plus filter as compared with both the minus and control filters on at
least two sets of autoradiograms exposed for different times. The
fourth lift was used to identify and eliminate phage for TIS10/PGS2 or
iNOS. After plaque purification, selected phage containing cloned
inserts were converted to Bluescript plasmids by in vivo excision using Exassist helper phage and the SOLR strain of E.
coli (Stratagene) as described by the manufacturer.
Amplification of Phage Inserts by
PCR
Plaque-purified candidate phage were replated once, picked
into 1 ml of water with 25 µl of chloroform, and stored frozen at
-20 °C. After thawing and refreezing twice, a 25-µl
aliquot was heated to 95 °C for 5 min and then used as template for
the PCR in a total volume of 50 µL, with 2.5 units of Ampli-Taq polymerase and the supplied buffer (Perkin-Elmer), dNTP
concentrations of 200 and 1 µM each of the primers
5`-CGGGCTGCAGGAATTC-3` and 5`-CCCCTCGAGTTTTTTTTTT-3`. These primers
were designed to hybridize just outside the 5` end of the insert at the EcoRI cloning site and at the overlap of the poly(A) tail of
the insert with the XhoI cloning site of Zap II,
respectively(15) . After an initial denaturation at 94 °C
for 3 min, reactions were cycled 30 times (15 s at 94 °C, 15 s at
48 °C, 1 min at 72 °C), with a final extension at 72 °C for
15 min. Products with a single band on agarose gel or PhastGel
(Pharmacia) electrophoresis were used as templates for random-primed
labeling with
P (Oligolabeling Kit, Pharmacia). If PCR was
unsuccessful or resulted in multiple bands, probes were obtained by
excision of the inserts from purified plasmids.
Sequencing
Sequencing of double-stranded plasmids
was performed at the Core DNA Sequencing Facility of the UCLA Jonsson
Comprehensive Cancer Center. Primers were designed using the Oligo
program (National Biosciences, Plymouth, MN). Sequences were assembled
and analyzed using the AssemblyLIGN and MacVector programs
(International Biotechnologies Inc., New Haven, CT).
The Strategy Used to Identify GARG
cDNAs
Pretreatment of Swiss 3T3 fibroblasts with TGF-1
augments induction by LPS of both TIS10/PGS2 and
iNOS(11, 16) . We sought to utilize this effect to
improve the sensitivity of screening for additional GARGs whose
induction by LPS might be similarly enhanced by TGF-
1. We
synthesized a cDNA library from poly(A)
-RNA extracted
from murine Swiss 3T3 fibroblasts pretreated with TGF-
1 and
stimulated for 2 or 4 h with either LPS or serum. Cycloheximide was
included during stimulation, so that only primary response genes would
be induced. We screened this cDNA library by differential
hybridization, using a plus cDNA probe synthesized from 3T3 cells
treated with both LPS and TGF-
1 and a minus probe from cells
treated with LPS and dexamethasone. Dexamethasone-suppressed cDNAs not
induced by LPS and/or TGF-
1 were eliminated by also requiring
enhanced hybridization with the plus probe versus a third
probe from untreated cells. TIS10/PGS2 and iNOS clones were identified
with specific probes, and eliminated. Because the minus probe treatment
did not include TGF-
1, our screen identified clones representing
either (i) GARG cDNAs, i.e. LPS-induced,
dexamethasone-attenuated messages whose induction by LPS might or might
not be augmented by TGF-
1 or (ii) TGF
-induced genes whose
message levels are not affected by dexamethasone. We confirmed the
differential expression of candidate clones and distinguished between
the GARG and non-GARG cDNAs by Northern analysis.
Cloning 12 GARG cDNAs and Two TGF-
We screened 15,000 plaques and selected 120 GARG cDNA
candidates. Forty-two phage demonstrating the greatest differences in
hybridization were chosen for initial evaluation. By
cross-hybridization with probes prepared by PCR from the cDNA inserts
of the plaque-purified phage, the 42-candidate phage were reduced to 10
independent groups. Northern analysis confirmed that eight of these
groups represented GARG cDNAs; the other two were not differentially
expressed. The 78 remaining phage yielded six additional candidate
groups. Northern analysis showed that four were GARG cDNAs, and two
were TGF-1-induced Non-GARG
cDNAs
1-induced genes not affected by dexamethasone. Thus, the
14 independent cDNAs that satisfied the screening criteria included 12
GARG and two TGF-
1-induced non-GARG cDNAs. Sequences from the ends
of the cDNAs were compared with nucleotide and protein data bases,
using BLASTN and BLASTX(17, 18) . We found that
seven of the GARG cDNAs and the two TGF-
1-induced non-GARG cDNAs
were known sequences; five of the GARG cDNAs had not been described
previously ().
Six of the Seven Known GARG cDNAs Encode Intercellular
Mediators
Six of the seven known GARG cDNAs encode secreted
proteins that function in intercellular communication. GARG-6/TSP1 is
thrombospondin-1, a secreted glycoprotein that modulates angiogenesis
and is chemotactic for
monocytes(19, 20, 21, 22) . LPS
induction of thrombospondin has not been described previously.
GARG-10/crg2 is the murine homologue of human IP-10, a CXC
chemokine that is chemotactic for monocytes and T
lymphocytes(23, 24, 25, 26) . GARG-13/JE
and GARG-17/MARC/fic are CC chemokines, the murine homologues of the
human monocyte chemotactic proteins MCP-1 and MCP-3,
respectively(27, 28, 29, 30, 31) .
GARG-33/MCSF or CSF-1 is murine macrophage-colony stimulating factor-1
(32). GARG-42/cyr61 is a growth factor-inducible immediate early gene
that encodes a member of a family of cysteine-rich secreted proteins
that act as growth regulators(33, 34, 35) . The
function of the seventh known GARG cDNA, GARG-49/IRG2, recently cloned
from an LPS-stimulated macrophage cell line, is unknown(36) .
Five GARG cDNAs Are Previously Undescribed
Five
GARG cDNAs represent previously undescribed genes. GARG-8, which
encodes a new CXC chemokine, is described below. GARG-16 and
GARG-39 encode distinct proteins related to GARG-49/IRG2 and to two
human interferon-inducible proteins of unknown function, IFI-56K and
ISG-54K (30-32). Thus, among the GARG cDNAs are three related but
distinct members of this family. Determination of the precise
relationship of GARG-16, -39, and -49 to each other and to the human
interferon-induced proteins awaits completion of sequencing. The
remaining two GARG cDNAs encode LPS-inducible, dexamethasone-attenuated
messages with no significant homology to any known genes.
GARG Messages Exhibit Diverse Patterns of Regulation in
Swiss 3T3 Cells
Message levels of all the GARGs are increased by
LPS and attenuated by dexamethasone ( Fig. 1and 2). However, the
GARG messages exhibit diverse responses to TGF-1 or serum.
TGF-
1 strongly induces most of the previously known GARGs (Fig. 1), but does not induce GARG-16, GARG-39, or GARG-49/IRG2.
Serum is a potent inducer of GARG-6/TSP1, GARG-33/MCSF, and
GARG-42/cyr61, but does not induce GARG-8/LIX, GARG-16, GARG-39, or
GARG-49. The modulating effects of TGF-
1 on serum or LPS induction
also vary for different GARGs. None of these GARGs exhibit the dramatic
augmentation of induction seen with TIS10/PGS2 and
iNOS(11, 16) . TGF-
1 modestly augments LPS
induction of GARG-33/MCSF and GARG-34. In contrast, TGF-
1 attenuates LPS induction of GARG-8/LIX, GARG-10/crg2, GARG-16,
GARG-39, and GARG-49/IRG2.
Figure 1:
The seven known GARG cDNAs: message
expression in Swiss 3T3 fibroblasts treated with dexamethasone,
TGF-1, serum, and LPS. Swiss 3T3 fibroblasts were grown to
confluence, serum-starved for 18 h, and then pretreated with 10 ng/ml
TGF-
1 for 1 h, 2 µM dexamethasone (DEX) for
3 h, or no pretreatment. After addition of serum (final concentration
20%), 10 ng/ml LPS, or no inducer, the cells were cultured for 4 more
hours and then harvested. Northern analysis was performed on replicate
panels from the same preparation of total cellular RNA (10
µg/lane). Each filter was probed with the constitutive control S2
to verify uniform loading; all were similar to the one shown (which
represents the filter used, sequentially, for GARG-49 and GARG-17).
Exposure times were: GARG-6/TSP1, 6 h; GARG-10/crg2, 16.5 h;
GARG-13/JE, 4.75 h; GARG-17/MARC, 16.5 h; GARG-33/MCSF, 9 h;
GARG-42/cyr61, 6 h; GARG-49/IRG2, 5 h; and S2, 5 h. Because exposure
times and probe lengths differ, the signal intensities of the different
cDNAs cannot be directly compared. The cell treatments corresponding to
the plus probe (solid triangle) and the minus probe (open
triangle) used in screening are
indicated.
Although induction of all the GARGs is
attenuated by dexamethasone, the degree of attenuation varies.
Dexamethasone almost completely suppresses LPS induction of
GARG-10/crg2 (>95%), substantially suppresses LPS induction of
GARG-8/LIX, GARG-13/JE, and GARG-17/MARC (75-80%) and modestly
attenuates LPS induction of GARG-34, GARG-42/cyr61, and GARG-61
(30-50%). For several GARGs, dexamethasone attenuation varies
with the inducer. Dexamethasone attenuates basal expression of
GARG-6/TSP1 by 87%, attenuates TGF-1 or LPS induction by
40-50%, but has a minimal effect (<10%) on serum induction.
Similarly, dexamethasone attenuates LPS induction of GARG-42/cyr61
(55%) and GARG-61 (35%), attenuates their TGF-
1 induction
(22-24%), but does not attenuate their serum induction.
Two Known TGF-
We cloned two cDNAs that are induced by
TGF-1-induced Non-GARG cDNAs Have
Intracellular Functions
1, LPS, and serum but are not attenuated by dexamethasone (, Fig. 3). TGF-
1-induced non-GARG (TnG) cDNAs
were an expected byproduct of our screening strategy: their induction
by TGF-
1 plus LPS is greater than their induction by LPS plus
dexamethasone, despite the absence of dexamethasone attenuation. Both
of the TnG cDNAs we cloned encode products with intracellular
functions. TnG-46/CEBP
encodes the CCAAT/enhancer-binding
protein-
, a transcription factor also known as NF-IL6 and CRP2
(37-40). TnG-54 encodes
-actin, a cytoskeletal protein.
Although
-actin message is abundant in unstimulated cells, its
expression is increased by growth factor
stimulation(41, 42) .
Figure 3:
The
two known TGF-1-induced, non-GARG cDNAs: message expression in
Swiss 3T3 fibroblasts treated with dexamethasone, TGF-
1, serum,
and LPS. Replicate filters from the same RNA preparations used in Figs.
1-2 were hybridized with
P-labeled probes and
autoradiographed for the indicated times: TnG-46/CEBP
(9 h),
TnG-54/
-actin (50 min), and S2 (6 h). The S2 panel shown
represents the filter used for TnG-54/
-actin. Loading on the other
filter (not shown) was similar.
Nucleotide Sequence and Deduced Amino Acid Sequence of
GARG-8
The GARG-8 cDNA sequence (Fig. 4) was derived from
two independent clones among the 120 primary candidates. The original
GARG-8 clone (nucleotides 13-1519 of the sequence in Fig. 4, followed by an 18-bp poly(A) tail) was completely
sequenced on both strands. A second clone (GARG-8.36) included 12
additional bp at the 5` end and a 120-bp poly(A) tail; it was sequenced
on both strands through the coding region and on the lower strand at
the 3` end. The GARG-8 cDNA has a 396-bp open reading frame starting
with ATG at nucleotide 66, which occurs in a context (CCACAATGA)
favorable for initiation of transcription according to the Kozak
rules(43, 44) . The predicted product, a 132-amino acid
peptide of 14,189 daltons, includes a hydrophobic stretch of 12 amino
acids (Met-22 through Leu-33) typical for a signal
peptide(45, 46) , followed by a candidate signal
peptidase cleavage site between alanines 40 and 41. The predicted
mature peptide has a mass of 9852 daltons. The open reading frame is
followed by a 1058-bp untranslated region that includes five ATTTA
sequences (arrows in Fig. 4A) and a repetitive
sequence (nucleotides 748-827) containing 40 repeats of AT or AG
(region R in Fig. 4A). The 3`-untranslated
region includes a segment with striking homology to sequences in other
genes (region H in Fig. 4A), as discussed
below. The polyadenylation signal AATAAA occurs at nucleotides
1499-1504.
Figure 4:
Sequence and translation of the GARG-8/LIX
cDNA. In the schematic diagram, A, the coding regions
corresponding to the predicted 40 amino acid signal sequence and 92
amino acid mature peptide are indicated by the open
rectangles. In the translated sequence, B, the alanine
that begins the predicted mature peptide is indicated by
&cjs1219;&cjs1219;&cjs1219;. The four conserved cysteines of the
CXC chemokine family are indicated in bold. The
3`-untranslated region includes five ATTTA sequences (arrows in A, bold in B), a repetitive sequence (R in A, dashed underline in B),
and a region with high homology (H in A, overline in B) to portions of the 3`-untranslated regions of two
other CXC chemokines. The poly(A) tail (dotted line in A, omitted in B) is preceded by a
polyadenylation signal (vertical line in A,
&cjs1219;&cjs1219;&cjs1219;&cjs1219;&cjs1219; in B).
The Predicted Product of the GARG-8 cDNA Is a New Member
of the CXC Chemokine Family
The predicted GARG-8 protein has
four cysteines in positions characteristic of CXC chemokines
(the first two being separated by one amino acid, Fig. 4) and
shares substantial homology (26-54% identical residues) with
known CXC chemokines (Fig. 5, ). We propose
the designation LPS-inducible CXC chemokine, or LIX, for this
predicted cytokine.
Figure 5:
Alignment of the LIX protein with selected
CXC chemokines. Representative members of the CXC
chemokine family, without the signal sequences, were individually
aligned with the predicted LIX protein. Amino acids identical to those
in the corresponding position in LIX are indicated by dots.
The four conserved cysteines are indicated by asterisks.
Abbreviations for species are: po, porcine; bov,
bovine; hu, human; mu,
murine.
Alignments of the LIX mature peptide with its
four closest relatives and a representative selection of other
CXC sequences are shown in Fig. 5. The COOH-terminal
region of LIX has a distinctive length and sequence. LIX extends
10-18 amino acids beyond the ends of all the other CXC
chemokines except for the very distantly related MIG peptides (). It should be noted that COOH-terminal lengths among
CXC homologues in different species are quite similar.
A Segment of the 3`-Untranslated Regions of LIX, AMCFII,
and ENA-78 Has Greater Nucleotide Homology Than Their Coding
Regions
A BLASTN search (17) of the nucleotide
data banks identified significant matches of LIX only with porcine
AMCFII and human ENA-78. Surprisingly, these nucleotide matches are
located not in the protein coding segments, but in the
3`-untranslated regions (3`-UTRs) of the mRNAs (Fig. 6A). A 125-bp segment of LIX (nucleotides
992-1126, designated H in Fig. 4A) contains a
40 nucleotide stretch (1) with 95% identity to the AMCFII 3`-UTR
and a 46 nucleotide stretch(1081-1126) with 96% identity to the
AMCFII 3`-UTR. In Fig. 6, all exactly conserved segments longer
than 11 nucleotides in either the 3`-UTRs (Fig. 6A) or
the coding regions (Fig. 6B) are indicated. In the H
segment of the 3`-UTR, there are stretches of 12, 25, 20, and 23
nucleotides exactly conserved between LIX and AMCFII and stretches of
12 and 16 nucleotides exactly conserved between LIX and ENA-78. In this
same portion of the 3`-UTRs, a 40-bp segment of AMCFII (948 to 987) has
95% homology to ENA-78 and includes stretches of 21 and 12 nucleotides
exactly conserved in AMCFII and ENA-78. In contrast, the protein-coding
regions of LIX, AMCFII, and ENA-78 do not include long exactly
conserved sequences (Fig. 6B). The longest segments
exactly conserved between the coding regions of LIX and AMCFII, and
between the coding regions of LIX and ENA-78, are only 11 nucleotides.
Figure 6:
Nucleotide conservation among murine LIX,
porcine AMCFII, and human ENA-78 in the conserved portions of their
3`-untranslated regions and in their protein coding regions.
Nucleotides in AMCFII and ENA-78 identical to those in LIX are
indicated by dots. In the homologous portions of the
3`-untranslated regions (A), segments longer than 11
nucleotides that are identical in LIX and AMCFII are indicated by the solid bar above the aligned sequences. The symbols below the
aligned sequences indicate segments longer than 11 nucleotides that are
identical in LIX and ENA-78 (dashed bar) or identical in
AMCFII and ENA-78 (diamonds). In the homologous portions of
the mature peptide coding sequences (B), there are no segments
longer than 11 nucleotides exactly conserved between LIX and AMCFII or
between LIX and ENA-78. A 13-nucleotide stretch exactly conserved
between AMCFII and ENA-78 is indicated (diamonds).
LIX Message Is Induced in Swiss 3T3 Cells by LPS and by
TGF-
LIX is induced in 3T3 cells by LPS
at concentrations as low as 0.1 ng/ml, with maximal induction (at 4 h)
achieved at 1 ng/ml (Fig. 7A). The other GARGs tested
also show maximal induction at 1-10 ng/ml. In response to LPS at
10 ng/ml, LIX expression peaks at 2-4 h, but remains well above
basal levels for at least 24 h (Fig. 7B). Serum produced
no detectable induction of LIX (Fig. 7C). In contrast,
three other chemokines, GARG-10/crg2, GARG-13/JE, and GARG-17/MARC, are
induced by serum ( Fig. 1and Fig. 7C). LIX is
transiently induced by TGF-1, but Not by Serum
1 (Fig. 7D); in
comparison, the responses of GARG-10/crg2 and GARG-13/JE to TGF-
1
are more sustained. Because different exposures were selected to best
illustrate the variations with time or LPS concentration, the relative
expression of GARG messages in Fig. 7, A-D, cannot
be compared directly. A quantitative comparison of LIX expression
following LPS, serum, or TGF-
1 stimulation is shown in Fig. 7E. Maximal TGF-
1-induced LIX expression is
17% of the maximal LPS-induced LIX expression.
Figure 7:
Induction of LIX and other GARG messages
in Swiss 3T3 cells. Serum-starved Swiss 3T3 fibroblasts were treated
with varying concentrations of LPS, with serum (20%), or with
TGF-1 (10 ng/ml), and harvested at 4 h (A) or at the
times indicated (B-E). Northern analysis of total
cellular RNA (10 µg/lane) was performed on duplicate filters, which
were probed with two or more cDNAs for messages of different sizes,
then stripped and reprobed. For each of the indicated cDNAs, the panels
shown in A-D are from autoradiograms of a single filter.
Loading on the duplicate filter, as indicated by the constitutive S2
probe, was similar to the one shown. The exposure times were: A, 6 h for TSP1, LIX, JE, S2; 14 h for crg2 and MCSF. B, same as in A except 6 h for MCSF; C and D, 14 h for crg2; 6 h for TSP1, JE, and MCSF; 9 h for LIX and
S2. E, quantitative comparison of LIX expression following
stimulation with LPS, serum, and TGF-
. The graph shows
the ratio of radioactivity in the LIX band to radioactivity in the S2
band for each lane, expressed as a percentage of the peak LIX/S2 ratio
observed at 2 h following LPS stimulation.
LIX Is a Primary Response Gene
Cycloheximide does
not block LPS induction of LIX (Fig. 8), demonstrating that LIX
is a primary response gene. Cycloheximide alone produces only a slight
increase in basal LIX expression and does not augment LPS-stimulated
LIX expression. Thus, LIX is not ``superinduced'' by
cycloheximide, unlike many primary response genes whose induced message
levels are markedly increased by inhibition of protein
synthesis(1) . Furthermore, addition of cycloheximide does not
affect the ability of dexamethasone to attenuate LPS induction of LIX.
Figure 8:
Effect of cycloheximide on LIX induction
by LPS and attenuation by DEX. Dexamethasone (2 µM) was
added to cultures of serum-starved Swiss 3T3 cells 3 h before addition
of 10 µg of cycloheximide (CHX), 10 ng/ml LPS, or both.
Cells were harvested 4 h later, and Northern analysis of total cellular
RNA (5 µg/lane) was performed. The filter was probed with P-labeled LIX and S2 cDNA, then stripped and reprobed with
JE. Exposure times were 8 h for LIX and S2 and 6 h for
JE.
In contrast to LIX, JE/GARG-13 message accumulation is induced by
cycloheximide alone, LPS-induced JE message is superinduced by
cycloheximide, and dexamethasone fails to attenuate LPS-induced JE
message accumulation in the presence of cycloheximide (Fig. 8).
The contrasting effects of cycloheximide on LIX and JE suggest that the
mechanisms controlling LPS induction and dexamethasone attenuation of
these two chemokines may differ.
LIX Message Expression Is Induced by LPS in Fibroblasts,
but Not in Macrophages
Swiss 3T3 cells have many fibroblast
characteristics. To determine if LIX is expressed in normal fibroblast
populations, we examined fibroblasts (passage three) cultured from
normal mouse embryos. Both LIX and JE are induced by LPS in early
passage mouse embryo fibroblasts (Fig. 9A). Because
activated macrophages are prominent producers of many
chemokines(50) , including GARG-10/crg2, GARG-13/JE, and
GARG-17/MARC, we expected LIX to be induced in these cells. However, we
were unable to detect LIX expression in RAW 264.7 macrophages (Fig. 9B), in J774.A.1 macrophages (not shown), in
peritoneal macrophages stimulated in vitro with LPS (not
shown), or in peritoneal macrophages after intraperitoneal injection of
LPS (Fig. 9C). Induction by LPS of JE or TIS10/PGS2 (not
shown) was readily detected in each case. These data suggest that LIX
is not produced by macrophages.
Figure 9:
LIX is
expressed in mouse fibroblasts, but not in macrophages. A,
Northern analysis of total cellular RNA (10 µg/lane) from mouse
embryo fibroblasts stimulated with LPS for 4 h. Filters were hybridized
with probes for LIX and S2 (17-h exposure), then stripped and reprobed
for JE (15 h). B, RAW 264.7 macrophages and Swiss 3T3
fibroblasts were stimulated with LPS (10 ng/ml) for 1-4 h, and
total cellular RNA (10 µg/lane) was analyzed. The filter was first
probed with LIX alone (43-h exposure), then with S2 (31 h) and JE (15
h). C, peritoneal macrophages were obtained by saline lavage
four hours after intraperitoneal injection of sterile saline (control)
or 25 µg of LPS. Cells from three mice in each group were pooled,
and total cellular RNA was isolated and analyzed (4 µg/lane). RNA
from Swiss 3T3 cells (10 µg/lane) was run on the same gel as a
positive control. The filter was probed with LIX and S2, then stripped
and reprobed with JE. Exposure times were 19 h for LIX and S2 and 15 h
for JE.
Glucocorticoid Attenuation Defines a Subclass of
Primary Response Genes
Although other mechanisms have been
proposed, it is now thought that the anti-inflammatory actions of the
glucocorticoids are largely due to their ability to attenuate the
induction of genes encoding critical inflammatory
regulators(2, 5, 10, 51) .
Glucocorticoid attenuation of such genes has typically been evaluated
retrospectively, following characterization of the gene or its product.
In this study, we cloned a group of cDNAs by screening specifically for
dexamethasone attenuation of LPS-induced messages. We identified twelve
GARG cDNAs from a screening of only 15,000 clones. Moreover, four of
these cDNAs were each represented by a single clone. These results
suggest that many LPS-induced GARGs have not yet been described. The
entire class of GARGs may be quite large, because it should also
include glucocorticoid-attenuated genes that are inducible by other
stimuli, but not by LPS. About half of the GARG genes we cloned are
strongly induced by TGF-1 alone or by serum alone. We would expect
that screenings for TGF-
1-induced GARGs, for serum-induced GARGs,
or for GARGs induced by other agents, will yield subsets of genes
overlapping but distinct from the LPS-induced subset of GARGs.
1. However, TGF-
1 did not have a major
modulating effect on LPS induction of most of the GARG genes we cloned
in this study (unlike TIS10/PGS2 and iNOS). Thus, it is likely that we
would have cloned most of the same cDNAs if we had omitted TGF-
1
from the cell treatment used for the plus probe and screened for
differential hybridization with ``LPS'' versus ``LPS + DEX'' probes instead of ``LPS +
TGF-
1'' versus ``LPS + DEX.''
However, we might not have cloned GARG-34, GARG-42/cyr61, and GARG-61
without the inclusion of TGF-
1, because their induction by LPS
alone is weak compared with their induction by LPS and TGF-
1
together. On the other hand, LPS induction of several GARG messages,
including LIX/GARG-8, was attenuated by TGF-
1. There may
well be other LPS-induced GARGs whose chances of being identified in
this screening were reduced by the inclusion of TGF-
1 in the cell
treatment used for the plus probe.
LIX/GARG-8 Is a Previously Unknown Member of the CXC
Chemokine Family
The LIX/GARG-8 cDNA is predicted to encode a
novel CXC chemokine. Unlike GARG-10/crg2, GARG-13/JE, and
GARG-17/MARC, which are all induced by serum in 3T3 fibroblasts and
induced by LPS in macrophages, LIX is neither induced by serum in 3T3
cells nor induced by LPS in macrophages. Porcine AMCFII, the closest
structural relative of LIX, is induced by LPS in
alveolar macrophages(47) . These regulatory differences,
together with its distinctive structural features, suggest that LIX is
a chemokine not previously described in any species. Further
investigation of the cell and tissue-specific pattern of LIX expression
in response to LPS and other inflammatory stimuli will be an important
component of future studies.
terminus, LIX contains an ELR sequence,
which correlates with neutrophil chemotactic activity among known
CXC members (50). The residues Leu-30, Val-32, Gly-36, and
Pro-37 in LIX are identical to corresponding amino acids in IL-8 that
have been implicated by mutational analysis as important for IL-8
receptor specificity and activity for
neutrophils(53, 54) . The presence of these residues and
the ELR motif suggests that LIX may have neutrophil chemotactic
activity. Several CXC chemokines are known to have multiple
activities(50, 55) ; this could also be true for LIX. It
will be interesting to determine if the long COOH-terminal region of
LIX affects its func-tional activity and whether the COOH-terminal and
NH
-terminal regions of the LIX protein undergo
post-translational processing.
LIX May Contain a Regulatory Sequence in the
3`-Untranslated Region
The 3`-UTR of the LIX message contains a
125-bp segment with strong homology to sequences in the 3`-UTRs of two
other CXC chemokines, porcine AMCFII and human ENA-78.
Remarkably, the nucleotide sequence identities in this untranslated
segment are much greater than in the protein coding regions of the LIX,
AMCFII, and ENA-78 messages, suggesting that this segment, conserved in
three species, serves an unknown regulatory function. An unusual
regulatory function has been noted for a seven-nucleotide motif located
in the 3`-UTR of GARG-13/JE: this seven-nucleotide motif is essential
for induction of transcription of JE in response to platelet derived
growth factor or serum(56) . Although this motif occurs in the
3`-UTRs of many other primary response genes, including CXC
chemokines of the gro/KC group, it is not present in LIX.
Glucocorticoid Attenuation of GARG Messages May Involve
Multiple Mechanisms
Glucocorticoids can cause transcriptional
repression via binding of the liganded glucocorticoid receptor to DNA
at a specific ``negative'' glucocorticoid response element or
at a composite cis-acting site, or via protein-protein interactions of
the liganded glucocorticoid receptor with other transcription
factors(5, 6, 61, 62, 63) .
Glucocorticoids also modulate mRNA levels by post-transcriptional
mechanisms(64) . In this study, we pretreated cells with
dexamethasone for three hours before induction with LPS or serum. Under
these conditions, secondary responses dependent on the synthesis of
downstream effectors could also occur. Although LIX and GARG-13/JE are
both primary response genes in 3T3 fibroblasts, the effects of
cycloheximide on LPS induction and dexamethasone attenuation are
different for these two chemokines. Together with the diversity of
responses to dexamethasone we noted among the GARG cDNAs, this
observation emphasizes that mechanisms of glucocorticoid attenuation
may vary both among different GARGs and for different inducers of a
single gene. In future studies, we will investigate the specific
mechanisms of dexamethasone attenuation of LIX and other GARGs and
evaluate their responses to a variety of natural glucocorticoid
hormones.
Table: GARG
and non-GARG cDNAs cloned in this study
Table: CXC chemokines:
amino acid identities with LIX and C-terminal lengths
/EMBL Data Bank with accession
number(s) U27267.
-inducible
protein-10; LPS, lipopolysaccharide; MCP-1 and -3, monocyte chemotactic
protein-1 and -3; MCSF, monocyte colony-stimulating factor; MIG,
monokine induced by interferon-
; PCR, polymerase chain reaction;
TGF-, transforming growth factor
1; TSP1, thrombospondin
1; 3`-UTR, 3`-untranslated region; bp, base pair(s).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.