©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Glucocorticoid-attenuated Response Genes Encode Intercellular Mediators, Including a New C-X-C Chemokine (*)

Jeffrey B. Smith (1), Harvey R. Herschman (2) (3) (4)(§)

From the (1)Division of Neonatology of the Department of Pediatrics, the (2)Departments of Biological Chemistry and Pharmacology, the (3)Molecular Biology Institute, and the (4)Laboratory of Structural Biology and Molecular Medicine, UCLA School of Medicine, Los Angeles, California 90095

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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.

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.


INTRODUCTION

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)()and nitric oxide synthase (iNOS) enzymes and cytokines such as JE/MCP-1 and KC/gro (1-3).

Induction of primary response genes encoding transcription factors involved in the G 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.

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.


EXPERIMENTAL PROCEDURES

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.

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.

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.).

Phage from the cDNA library were plated at a density of 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).


RESULTS

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-1-induced Non-GARG cDNAs

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 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-1-induced Non-GARG cDNAs Have Intracellular Functions

We cloned two cDNAs that are induced by TGF-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.

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.

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-1, but Not by Serum

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 (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.




DISCUSSION

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.

In addition to glucocorticoid attenuation, our screening strategy involved pretreatment with TGF-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.

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.

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.

The function of LIX is unknown. Near its predicted NH 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.

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.

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.

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.

  
Table: GARG and non-GARG cDNAs cloned in this study

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.


  
Table: CXC chemokines: amino acid identities with LIX and C-terminal lengths

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.



FOOTNOTES

*
This work was supported by the National Institutes of Health Grant GM24797 and by Contract DE FC0387ER60615 between the Regents of the University of California and the Department of Energy. The UCLA DNA Core Sequencing Facility is funded in part by National Cancer Institute Jonsson Comprehensive Cancer Center Core Grant Award CA16042. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U27267.

§
To whom correspondence and reprint requests should be addressed: Dept. of Biological Chemistry, UCLA Center for the Health Sciences, Los Angeles, CA 90095. Tel.: 310-825-8735; Fax: 310-825-1447.

The abbreviations used are: PGS2, prostaglandin synthase 2; AMCFII, alveolar macrophage chemotactic factor II; CEBPß, CCAAT/enhancer-binding protein-ß; DEX, dexamethasone; ENA-78, epithelial neutrophil activating peptide-78; FBS, fetal bovine serum; GARG, glucocorticoid attenuated response gene; GCP2, granulocyte chemotactic peptide-2; IL-8, interleukin-8; iNOS, inducible nitric oxide synthase; IP-10, interferon--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).


ACKNOWLEDGEMENTS

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.


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