Cell Migration and Invasion Secretoglobin That Regulates Chemotactic Stimulates the Expression of a Novel Γ

The Journal of Immunology

IFN-␥ Stimulates the Expression of a Novel Secretoglobin That Regulates Chemotactic Cell Migration and Invasion

Moonsuk S. Choi, Rabindranath Ray, Zhongjian Zhang, and Anil B. Mukherjee1 IFNs are a family of cytokines that alert the immune system against viral infections of host cells. The IFNs (IFN-␣, IFN-␤, and IFN-␥) interact with speciﬁc cellular receptors and stimulate the production of second messengers, leading to the expression of antiviral and immunomodulatory proteins. We report in this study that IFN-␥ stimulates the expression of a novel gene that encodes a protein with 30% amino acid sequence identity with uteroglobin, the founding member of the newly formed Secreto- globin (SCGB) superfamily. We named this protein IFN-␥-inducible SCGB (IIS), because its expression in lymphoblast cells is augmented by IFN-␥ treatment. IIS is expressed in virtually all tissues, and the highest level of expression is detectable in lymph nodes, tonsil, cultured lymphoblasts, and the ovary. Interestingly, although the expression of IIS mRNA is not signiﬁcantly different in resting lymphoid cells, it is markedly elevated in activated CD8؉ and CD19؉ cells. Furthermore, treatment of lymphoblast cells with IIS antisense phosphorothioate (S)-oligonucleotides prevents chemotactic migration and invasion. Taken together, these results raise the possibility that this novel SCGB has immunological functions. The Journal of Immunology, 2004, 172: 4245Ð4252.

potent cytokine, IFN-␥ mediates a wide range of immu- Moreover, several paralogous proteins in different mammalian nological activities, primarily in response to viral infec- species have also been described. These paralogous proteins in- A tions or to inflammatory stimuli (1Ð4). In addition, IFNs clude the rat prostatic binding protein (20) also known as pros- (i.e., IFN-␣, IFN-␤, and IFN-␥) may regulate the amplification of tatein (21) or prostate-␣ protein (22). Prostatein forms oligomers Ag presentation to specific T cells and are expressed constitutively among its subunits C1/C3 and C2/C3, and is reported to inhibit by most cells. The physiological functions of IFNs are mediated microtubule assembly (23). Several homologous proteins to rat through both autocrine and paracrine mechanisms. prostatein have been described in other species. The rat prostatein The type I (␣ and ␤) and type II (␥) IFNs regulate overlapping C3 shows 34 and 41% sequence identity with human lacryglobin sets of several hundred genes at the transcriptional level (3, 5, 6) (24) and mammaglobin (MGB)1 (25), respectively. The protein (supplemental data for Ref. 3 at http://arjournals.annualreviews.org/ sequence of lacryglobin has been reported to be identical with that doi/suppl/10.1146/annurev.immunol.15.1.749). IFN-␥ also stimulates of MGB2 (26) and lipophilin (LIP)-C (27). Although the biological the expression of uteroglobin (UG),2 the founding member of the functions of most proteins in this superfamily remain unclear, UG newly formed Secretoglobin (SCGB) superfamily of proteins (7) that has been recognized as a multifunctional protein with potent anti- manifests potent anti-inflammatory and immunomodulatory proper- inflammatory, antichemotactic, and tumor suppressor-like activities (reviewed in Ref. 8). Blastokinin (9) or UG (10), first isolated ties (8). MGB1 is reported to be expressed at high levels in mam- from the uterus of rabbits during early pregnancy, is a homodimeric mary tumors (28). Interestingly, among the proteins of the SCGB protein in which the identical 70-aa subunits are connected in antipa- superfamily, the expression of only the UG gene has been reported rallel orientation by an N-terminal and a C-terminal disulfide bond to be stimulated by IFN-␥ (14, 29, 30). Thus, it is of interest to forming a central hydrophobic cavity. Although the functional signif- determine whether the expression of other members of this super- icance of this central cavity is unclear, it is suggested that it may family of genes are inducible by cytokines. sequester hydrophobic ligands such as progesterone, retinol, and poly- In this study, we report the characterization of the cDNA and the chlorinated biphenyls (11Ð13). gene encoding a novel SCGB, the expression of which in lympho- During the past two decades, the isolation and characterization blasts is stimulated by IFN-␥, and we named this protein IFN-␥- of cDNAs encoding UG from the mouse (14), pig (15), Syrian inducible SCGB (IIS). This protein bears 30% amino acid se- hamster (16), horse (17), and human (18, 19) have been reported. quence identity with UG, a multifunctional protein with potent anti-inflammatory/immunomodulatory properties. We also found that IIS is expressed in virtually all tissues, and although the ex- Section on Developmental Genetics, Heritable Disorders Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD pression of IIS mRNA is not significantly different in resting lym- 20892 phoid cells, it is markedly elevated in activated CD8ϩ T cells and ϩ Received for publication October 27, 2003. Accepted for publication January CD19 B cells. Moreover, treatment of the lymphoblast cells with 22, 2004. IIS antisense phosphorothioate (S)-oligonucleotide markedly in- The costs of publication of this article were defrayed in part by the payment of page hibited chemotactic migration and invasion, raising the possibility charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. that IIS may have immunological functions. 1 Address correspondence and reprint requests to Dr. Anil B. Mukherjee, Section on Developmental Genetics, Heritable Disorders Branch, National Institute of Child Materials and Methods Health and Human Development, National Institutes of Health, Building 10, Room Materials 9S241, Bethesda, MD 20892-1830. E-mail address: [email protected] 2 Abbreviations used in this paper: UG, uteroglobin; SCGB, secretoglobin; MGB, Polyclonal Ab of IIS was raised in the rabbit (Covance Laboratories, Vi- mammaglobin; LIP, lipophilin; IIS, IFN-␥-inducible SCGB; SSCP, single-strand con- enna, VA) against a synthetic oligopeptide (NH2-SFKKRLSLKKSWWK- formation polymorphism; SNP, single nucleotide polymorphism; STRP, short tandem COOH) corresponding to the amino acid sequence of IIS (residues 70Ð83) repeat polymorphism; IP-10, IFN-␥-inducible protein-10; S, phosphorothioate. (Peptide Technologies, Gaithersburg, MD). Specificity of this antiserum

toward IIS was also tested before and after adsorption with the oligopeptide TGA TTT ATT AAA GC-3Ј, annealed at 58¡C. PCR products were sub- that was used as the immunogen for raising this Ab. The results showed cloned into TOPO TA vector (Invitrogen). The nucleotide sequence was that, although unadsorbed antiserum readily recognized IIS protein band in determined by using a Beckman Coulter (Fullerton, CA) CEQ 2000 fol- Western blots, the postadsorbed antiserum was ineffective in recognizing lowing the manufacturer’s protocol. To investigate the tissue-specific ex- this protein. Human recombinant cytokines (IFN-␥, IL-4, and IL-13) and pression, PCR was also performed with first-strand cDNAs from various TNF-␣ were obtained from Sigma-Aldrich (St. Louis, MO) and Endogen human tissues as supplied in MTC panel II (Clontech) using the same (Woburn, MA), respectively. Genomic DNAs of 34 unrelated individuals primers and conditions mentioned above. PCR products were resolved by were obtained from the DNA Polymorphism Discovery resources of Co- electrophoresis using 1.2% agarose gels. The amplification of GAPDH was riell Cell Repositories (Camden, NJ). used to normalize cDNA loading into each lane. Cell culture Characterization of RT-PCR products by hybridization with IIS Immortalized normal human lymphoblast cells were obtained from Dr. K. cDNA probe Wisniewski (New York State Institute for Basic Research, Staten Island, Complimentary DNA from various human tissues (MTC panel II; Clon- N.Y.). These cells were grown in RPMI 1640 (Invitrogen, San Diego, CA) tech) were subjected to PCR as described above, and the products were supplemented with 16% heat-inactivated FBS (Invitrogen), 1 mM L-glu- ␮ resolved by electrophoresis using 1% agarose gels, and cDNA bands were tamine, and penicillin (100 U/ml)-streptomycin (100 g/ml), and incubated transferred on to Hybond-Nϩ nylon membrane (Amersham Biosciences, in a humidified incubator in an atmosphere of 5% CO and 95% air at 2 Buckinghamshire, U.K.). The blot was hybridized with [␣-32P]dCTP-la- 37¡C. Human colon carcinoma (T-84; ATCC no. CCL248; American Type beled IIS cDNA probe at 42¡C overnight using DIG Easy Hyb (Boehringer Culture Collection, Manassas, VA) and the NIH 3T3 cells were grown in Mannheim, Mannheim, Germany). After prehybridization for 2 h, the blot Dulbecco’s modified minimal essential medium (Invitrogen) supplemented was washed three times with SSC (2ϫ)/0.1% SDS solution for 15 min each with 10% heat-inactivated FBS (Invitrogen), 1 mM L-glutamine, and pen- ϫ ␮ and then three times with SSC (0.1 )/0.1% SDS solution for 15 min each. icillin (100 U/ml)-streptomycin (100 g/ml), and incubated in a humidified Autoradiographs were prepared using BioMax x-ray film (Kodak). atmosphere with 5% CO2 at 37¡C. Single-strand conformation polymorphism (SSCP) Expression of IIS protein in COS-1 cells To detect mutations (polymorphism), we carried out SSCP. Five fragments cDNA containing the entire coding sequence of IIS was amplified using Ј Ј Ј of IIS gene were amplified from genomic DNAs of normal individuals (n ϭ 5 -ATG AGG CTG TCA GTG TGT CTC C-3 (forward) and 5 -TCA Ј 34) by PCR solution added [␣-32P]dCTP. The fragment (382 bp) of pro- CTA TTT CCA CCA GGA CT-3 (reverse) primers, and cDNA encoding moter region 1 was amplified using 5Ј-GTG GCT ACA CAT CAC AGA the mature IIS (without the signal peptide sequence) without signal peptide Ј AAG-3Ј (forward) and 5Ј-CAC AGG TGA ATT ATG GCT TC-3Ј (re- sequences was amplified using 5 -CTT GTC TGC CCA GCT GTT GCT Ј Ј Ј verse) primers at annealing temperature of 64¡C. The fragment (398 bp) of TC-3 (forward) and 5 -TCA CTA TTT CCA CCA GGA CT-3 (reverse) promoter region 2 was amplified using 5Ј-GAG AAC ACA GCC TTC primers and subcloned into pcDNA4/HisMax-TOPO TA expression vector CAG C-3Ј (forward) and 5Ј-CAA TGA GTG ATT TGG ATT CG-3Ј (re- (Invitrogen). The recombinant plasmids were linearized with BglII and verse) primers at annealing temperature of 63¡C. The fragment (160 bp) of electroporated into COS-1 cells. The transfected cells were culture for 48 h exon 1 was amplified using 5Ј-CTC CAT GAC TAC ACA GGC TC-3Ј in Dulbecco’s modified minimal essential medium containing 10% FBS, ␮ (forward) and 5Ј-GCT GGA CTC ATG ACT GAT G-3Ј (reverse) primers 0.8 mM L-glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin. at annealing temperature of 65¡C. The fragment (311 bp) of exon 2 was amplified using 5Ј-CTG TCT GGT GTA ACC TCA GG-3Ј (forward) and Expression of rIIS protein in Escherichia coli 5Ј-GCT GAG TTG AAT TCT GCC TC-3Ј (reverse) primers at annealing IIS cDNA containing the entire coding region was amplified using 5Ј-ATG temperature of 65¡C. The fragment (210 bp) of exon 3 was amplified using AGG CTG TCA GTG TGT CTC C-3Ј (forward) and 5Ј-CAT TTT TTC 5Ј-CAG CAG CAG CAT GAC TGA C-3Ј (forward) and 5Ј-GAC CAG ACT ATT TCC ACC AGG ACT-3Ј (reverse) primers and subcloned into TGG AGA TGT GCA G-3Ј (reverse) primers at annealing temperature of PshI/EcoRV site of pET-42a(ϩ) vector (Novagen, Madison, WI), which 66¡C. The amplified fragments were resolved on 0.5ϫ MDE gel (BioWhit- includes an integrated T7 promoter. E. coli strain, BL21 (DE3) was trans- taker Molecular Applications, Rockland, ME) with 0.6ϫ Tris-borate- fected with the cDNA construct, and the transformed cells were inoculated EDTA (TBE) running buffer at constant 3 W for 12Ð16 h, and autoradio- into Luria-Bertani broth with kanamycin and grown until it reached an OD graphs were obtained. Thermo sequenase radiolabeled terminator cycle reading of 1.0 at 600 nm. Expression of the IIS protein was induced with sequencing kit (USB, Cleveland, OH) was used for the sequencing follow- 0.4 mM isopropyl-␤-D-thiogalactopyranoside for 2 h. ing the manufacturer’s protocol. The samples were resolved by electrophoresis using 6% sequencing gel, and autoradiographs were prepared by Purification of rIIS protein from the transfected E. coli culture using BioMax x-ray film (Kodak, Rochester, NY). IIS protein from the transfected E. coli lysates was purified using Bug- RT-PCR Buster Ni-NTA His ⅐ Bind purification kit and His ⅐ Bind columns (Nova- gen). The transfected E. coli cells (1.0 g of cell paste) were harvested and Total RNAs were extracted from human lymphoblast and human colon added with 5 ml of BugBuster reagent and 5 ␮l of Benzonase (Novagen) carcinoma cells using TRIzol reagent (Invitrogen) following the manufac- followed by incubation for 20 min on a shaking platform. The solution was turer’s instructions. Total RNAs were reverse-transcribed using Thermo- centrifuged, and the supernatant was passed through a 0.45-␮m syringe- Script RT-PCR system (Invitrogen). Semiquantitative PCR was performed end filter and then loaded onto His ⅐ Bind column precharged with Ni2ϩ. to amplify target fragments of cDNA. Five microliters of the RT-PCR The column was washed with 10 vol of 1ϫ binding buffer and 6 vol of 1ϫ product was used for the PCR. The PCR conditions are as follows: dena- washing buffer. The bound protein was eluted with 6 vol of 1ϫ elution turation at 94¡C for 1 min, followed by 35 cycles of amplification at 94¡C buffer. for 30 s and at different annealing temperature for 2 min, and a final incubation at 68¡C for 5 min using AdvanTaq Plus DNA polymerase (Clon- Immunoprecipitation and immunoblotting tech, Palo Alto, CA). The primers that amplify LIP-B and an unknown cDNA fragment are 5Ј-CTG CTG CTA CCA GGC CAA TG-3Ј (forward) Human lymphoblasts were harvested and lysed with lysis buffer containing and 5Ј-GTC ACA CAC TAC ATT TCT TC-3Ј (reverse), and the primers protease inhibitor mixture and immunoprecipitated using protein A immu- that amplify only LIP-B are 5Ј-CCT CTG TTC AAG TTA AGT C-3Ј noprecipitation kit (Kirkegaard & Perry Laboratories, Gaithersburg, MD) (forward) and 5Ј-CCG CAA TGA GGC TTC GTT TGG-3Ј (reverse). The according to the supplier’s instructions. The cell lysate was precleared annealing temperature is 60¡C. The primers used to amplify MGB1 were using 50% resin slurry. The IIS Ab was added to the precleared sample 5Ј-GAC AAT GCC ACT ACA AAT GCC-3Ј (forward) and 5Ј-CAT TGC followed by an addition of 50 ␮l of 50% resin slurry and incubated over- TCA GAG TTT CAT CCG-3Ј (reverse) with annealing temperature of night at 4¡C with gentle agitation. The resin was pelleted and washed three 66¡C. The primers used for the amplification of other cDNAs are as fol- times with lysis buffer and resuspended in 40 ␮lof1ϫ SDS-PAGE sample lows: MGB2, forward, 5Ј-CTC CTG GAG GAC ATG GTT G-3Ј, reverse, buffer. Proteins were resolved by electrophoresis on 18% Tris-glycine gels 5Ј-CTA TGT GAC TGG TTG AGG-3Ј, annealing temperature 66¡C; (Invitrogen). Total proteins from transfected COS-1 cells and transfected LIP-A, forward, 5Ј-CAG TGG TCT GCC AAG CTC TTG G-3Ј, reverse, E. coli cells were extracted using T-PER mammalian and bacterial protein 5Ј-CAT AGG CCA TCG TAT CCA CGC-3Ј, annealed at 66¡C; UG, for- extraction buffers (Pierce, Rockford, IL), respectively, according to the ward, 5Ј-CAG AGA TCT GCC CGA GCT TTC-3Ј, reverse, 5Ј-GCT TAA manufacturer’s instructions. The concentration of total proteins was deter- TGA TGC AAA CAC TGG-3Ј, annealed at 58¡C; IIS, forward, 5Ј-CTC mined using Bradford protein assay (Bio-Rad Laboratories, Hercules, CA). ACA GCC GAA TAA GCC ACC-3Ј, reverse, 5Ј-GTG CAG GGC AAG One hundred micrograms of total protein from the transfected COS-1 cells The Journal of Immunology 4247 were resolved by electrophoresis using 18% Tris-glycine gel (Invitrogen) using the cell invasion assay kit as indicated above. The migrated and followed by electrotransferring onto Immobilon-P transfer membrane (Mil- invaded cells were counted using a hemocytometer. lipore, Bedford, MA). The blots were incubated in blocking solution containing 4% BSA at 4¡C. They were washed with TBST solution (10 mM Statistical analysis Tris-HCl (pH 8.0), 0.15 M NaCl, and 0.1% Tween 20) and incubated with anti-Xpress Ab (diluted 1/5000; Invitrogen) for 1 h followed by incubation The statistical significance was analyzed using Student’s t test. Data are presented as the mean Ϯ SD. The results were considered significant at with HRP-conjugated rabbit anti-mouse Ab. The blots were washed with Ͻ TBST solution and developed using ECL chemiluminescence detection kit p 0.05. (ECL kit; Amersham Biosciences) according to manufacturer’s instructions. The purified proteins from the transfected E. coli culture were re- Results solved on 10% Tris-glycine gel followed by transferring onto Immobilon-P IIS is a novel member of the SCGB family transfer membrane. The membrane was incubated in blocking solution containing 1% gelatin at 4¡C. The blot was washed and incubated with alkaline To delineate the levels of expression of various members of the phosphatase-conjugated S ⅐ Tag Ab (diluted 1/5000; Novagen) for 15 min. SCGB family in human lymphoid cells, we performed RT-PCR The blots were washed with TBST solution and developed using S ⅐ Tag using total RNA from a lymphoblast cell line, using primers for AP Western blot kit (Novagen) following the manufacturer’s instruction. UG, LIP-A, LIP-B, MGB1, and MGB2 (Fig. 1A). We performed For detecting the IIS protein bands, the blots were incubated with anti-IIS Ab (diluted 1/1000) for 1 h, washed with TBST solution, and incubated DNA sequencing of the PCR products to confirm the identity of with HRP-conjugated goat anti-rabbit IgG (diluted 1/1000; Amersham Bio- each of these genes. Unexpectedly, we found that the amplified sciences) for 1 h, washed five times (10 min each), and detected by ECL products for putative LIP-B contained a DNA sequence that is chemiluminescence detection. The specificity of the Ab was determined by unrelated to that of LIP-B. LIP-B cDNA and the unidentified RT- preabsorption of the antiserum with the synthetic oligopeptide Ag, and this postadsorbed antiserum failed to recognize the IIS protein band in Western PCR product were independently amplified by PCR using more blot. The prestained protein marker (Bio-Rad Laboratories) was used as a specific primers for LIP-B and a separate set of primers for the molecular mass standard. unique sequence. The PCR products were analyzed by DNA se- Quantitative real-time RT-PCR quencing. The results of repeated experiments showed that not only all members of the SCGB gene family but also the putative Cultured human lymphoblast cells were treated with the following cyto- new gene is also expressed in human lymphoblasts (Fig. 1A). kines: TNF-␣ (10 ng/ml), IFN-␥ (10 ng/ml), IL-4 (0.5 ng/ml), or IL-13 (10 ng/ml) for 1, 3, and 6 h, respectively. The cells were also treated with To further delineate the identity of this unique RT-PCR product, different concentrations of IFN-␥ (0.1, 1, or 10 ng/ml) for1htoobtain a we performed a BLAST search of GenBank database and uncov- dose-response curve. Total RNA was isolated from the treated cells using ered that the sequence of the unidentified RT-PCR product is iden- TRIzol (Invitrogen) and an RNeasy mini-kit (Qiagen, Valencia, CA), fol- tical with that of the expressed sequence tag 378247, MAGE se- lowed by DNase treatment to eliminate genomic DNA contamination. quences, and MAGI Homo sapiens cDNA sequence (GenBank Quantitative real-time RT-PCR was performed using Smart Cycler system (Cepheid, Sunnyvale, CA). First-strand cDNA was synthesized from 1 ␮g accession no. AW966174). Interestingly, we were also able to am- of total RNA using Superscript III first-strand synthesis system (Invitro- plify this cDNA by RT-PCR using total RNA from human colon gen) following the manufacturer’s protocol. Real-time PCR was performed carcinoma cells. Further analysis of the full-length cDNA se- with 2.5 ␮l of cDNA and primers (forward, 5Ј-CTC ACA GCC GAA TAA quence contained a single open reading frame of 252 nt. Because GCC ACC-3Ј; reverse, 5Ј-GTG CAG GGC AAG TGA TTT ATT AAA GC-3Ј) using QuantiTect SYBR Green (Qiagen) following the manufac- this unique sequence has not been previously described and be- turer’s protocol under the following conditions: denaturation at 94¡C for 15 cause the expression of this gene is stimulated by IFN-␥,we min followed by 50 cycles of amplification at 94¡C for 15 s, 58¡C for 30 s, named this gene IIS. The cDNA sequence of IIS has been submit- and 72¡C for 30 s. Real-time PCR was also performed with first-strand ted to GenBank (accession no. AY236538). The IIS cDNA en- cDNAs from resting and activated blood cells using Human Blood Frac- codes a protein of 83 aa, which has a calculated molecular mass of tions MTC panel (Clontech). The data from each PCR run was analyzed using Cepheid Smart Cycler software program (Cepheid) with FAM as the 9.2 kDa and a calculated isoelectric point of 8.9. reference dye. The final data were normalized to ␤-actin and are presented as fold induction. Quantitation was performed using at least three separate total RNA samples for each treatment group. Antisense S-oligonucleotide treatment A phosphorothioate IIS antisense oligonucleotide (5Ј-TTT GGC AAC TTG GAG GTT TA-3Ј) was used to inhibit IIS protein expression. The corresponding IIS sense oligonucleotide (5Ј-TAA ACC TCC AAG TTG CCA AA-3Ј) was used as one of the controls. When the lymphoblast cells reached 30Ð40% confluence, they were rinsed once with serum-free Opti- MEM I (Invitrogen). The S-oligonucleotides were then delivered to the cells by Oligofectamine (Invitrogen) according to the manufacturer’s instructions. Treatment of the cells with Oligofectamine alone was also used as a control. The cells were incubated for 60 h at 37¡C before using for migration and invasion assays. Cell migration and invasion assay The cell migration assay was performed using QCM 96-well 5-␮m cell migration plates (Chemicon, Temecula, CA). The cells were collected after transfection with IIS antisense or sense S-oligonucleotides and incubated FIGURE 1. The mRNA expression of SCGB family members in human for 60 h. Following incubation, the cells were rinsed with serum-free RPMI lymphoblast cells, the gene structure of IIS, and exon/intron boundaries. A, 1640 medium and resuspended in the same serum-free medium. One hun- The mRNA expression levels of SCGB family members in human lym- dred fifty microliters of the conditioned medium from NIH 3T3 cell culture phoblast cells by semiquantitative RT-PCR. Lane 1, UG; lane 2, LIP-A; ϫ 5 ␮ was placed into 96-well feeder tray, and 1 10 cells in 100 l of serum- lane 3, MGB1; lane 4, MGB2; lane 5, LIP-B; lane 6, IIS. B, Schematic free RPMI 1640 medium was placed into cell migration chamber plate and structure of the human IIS gene with the exons shown by boxes and the then incubated for 24 h at 37¡CinaCO2 incubator. The cell invasion assay was performed using QCM 96-well cell invasion plate (Chemicon). The introns shown by horizontal lines. The coding region is shown by solid transfected cells were rinsed with serum-free RPMI 1640 medium and then boxes. There is an STRP in intron 2 and an SNP in the 3Ј region of the resuspended in serum-free RPMI 1640 medium. The conditions for the gene. C, Exon-intron boundaries of the human IIS gene. The consensus invasion assay are identical with those of the migration assay conducted nucleotides of exon-intron boundaries are in bold. 4248 IFN-␥-INDUCIBLE SCGB

BLAST search against the human genome database using the IIS C(T)9, or (CTTT)2(CTTTT) in intron 2 were detected, and this cDNA coding sequence enabled us to identify the genomic se- polymorphism appears to occur at frequencies of 0.662 for C(T)8, quence containing the IIS gene. We found that the entire IIS gene 0.309 for C(T)9, and 0.029 for (CTTT)2(CTTTT) in these individ- sequence was contained in human chromosome 11, clones RP11- uals (Fig. 2C). 703H8 (GenBank accession no. AP003306), pDJ741n15 (accession no. AC004127), and CTD-253 D15 (accession no. IIS mRNA expression in various tissues AP003064). To determine IIS gene structure, we further analyzed We performed Northern hybridizations using poly(A)ϩ RNA blot these clone sequences and found that the entire IIS gene spanned of various tissues to see the level of IIS mRNA expression. How- ϳ3 kb of genomic DNA and a total of three exons and two introns ever, this approach was unsuccessful, because the signal from (Fig. 1B). The sequences adjacent to the splice sites were in good these tissues was extremely low (data not shown). Therefore, we conformance with the consensus splice rule (Fig. 1C). The relative determined the expression of IIS mRNA by semiquantitative RT- sizes of the exons and introns are closely conserved in SCGB PCR. The identities of the RT-PCR products were confirmed by family members except for exon 3 of IIS, which encodes the short- Southern hybridization using IIS cDNA probe as well as by DNA est amino acid sequences among the human SCGB members. Sub- sequencing. The results show that the expression of IIS mRNA is sequent BLAST analysis demonstrated that cDNA sequence of IIS the highest in the ovary, lymph node, and tonsil, and in a lympho- also showed high homology to those of other SCGB family mem- blast cell line. Moderate expression levels were detected in the bers, especially with LIP-B cDNA sequences showing a 72.6% small intestine, colon, bone marrow, and fetal liver, and in a colon homology. carcinoma cell line, T-84, and the lowest expression levels were The IIS gene promoter sequence was analyzed using NSITE DB detected in the spleen, thymus, prostate, and the testis (Fig. 3). program to determine the transcription factors that potentially regulate this gene. Several transcription factor-binding consensus se- IIS protein expression in bacterial and mammalian cells quences including those of NF-␬B, hepatocyte NF-1A, -1B, -1C, IIS cDNA was subcloned into PshI/EcoRV site of pET-42a(ϩ) SP1, IFN-stimulated response elements, and ␥-IFN-activated sites vector, and the expression constructs were electroporated into E. Ϫ are present within 1500 bp of the IIS gene promoter region. coli strain BL21 (DE3). After induction with isopropyl-␤-D-thiogalactopyranoside, IIS protein was purified using BugBuster Ni- Polymorphisms in the IIS gene NTA His ⅐ Bind purification kit and His ⅐ Bind columns. The eluted Because single nucleotide polymorphisms (SNP) have been re- protein (molecular mass, 35 kDa) was detected with S ⅐ Tag Ab ported in the UG gene (19), we sought to determine whether SNPs and with IIS Ab (Fig. 4A). These results show that IIS mRNA is are also detectable in the IIS gene. IIS genomic DNAs from 34 translated to IIS protein in the bacteria, and that it is stable in the normal healthy individuals were analyzed for polymorphism by bacteria. SSCP and DNA sequencing. There were five different SSCP pat- We also expressed the IIS cDNA in COS-1 cells. The IIS cDNA terns identified in amplicons of the exon 3 region (Fig. 2A). We was subcloned into pcDNA4/His ⅐ Max-TOPO vector and was found an SNP (G3A substitution) in the 3Ј-flanking region near electroporated into COS-1 cells. The lysates of the transfected cells exon 3 (Fig. 2B). The frequency of occurrence of this SNP was were resolved by SDS-PAGE, and protein bands were analyzed by 0.971 for the G allele and 0.029 for the A allele (n ϭ 34 individ- Western blot. The results show that IIS protein was stably ex- uals). Short tandem repeat polymorphisms (STRP) such as C(T)8, pressed in COS-1 cells both without (Fig. 4B, lane 2) and with the signal peptide (lane 3). No change in the apparent molecular mass of this protein was observed when we resolved the protein by SDS-PAGE under both reducing and nonreducing conditions (Fig. 4B), suggesting the absence of subunit structure involving inter- chain disulfide bonds. Using IIS Ab and immunoprecipitation, we further confirmed that a cultured human lymphoblast cell line ex- presses IIS protein (Fig. 4C, upper panel). The specificity of the IIS Ab was determined by adsorbing the total antiserum with the synthetic oligopeptide corresponding to the IIS amino acid sequence (residues 70Ð83), which failed to recognize the protein band (Fig. 4C, lower panel).

FIGURE 2. Polymorphism of IIS gene. A, SSCP analysis of IIS genomic DNA. Each lane shows different SSCP pattern from normal in- FIGURE 3. The mRNA expression levels of IIS in various human tis- dividuals. B, SNP (G3A) of IIS gene in 3Ј region of the gene shows three sues and cell lines. The mRNA expression levels were detected by RT-PCR different genotypes: GG (left panel), GA (middle panel), and AA (right and then Southern hybridization. Note the highest level of expression of IIS panel). Arrows indicate SNP sites. C, There is STRP (C(T)8, C(T)9,or mRNA in the ovary, lymph node, and tonsil, and in lymphoblasts. The (CTTT)2(CTTTT)) of IIS gene in intron 2. Panels show C(T)8/C(T)8 (left), ampliﬁcation of GAPDH was used to normalize the total RNA added to C(T)9/C(T)9 (middle), and (CTTT)2(CTTTT)/C(T)8 (right) genotypes. each RT-PCR. The Journal of Immunology 4249

FIGURE 4. Expression of IIS protein. A, Immunoblot analysis of rIIS expressed in E. coli. IIS protein was detected with S ⅐ Tag Ab (left panel) and IIS Ab (right panel) following the purification using Ni2ϩ resin. Lane 1, Lysate from the cells transfected with vector alone; lane 2, lysate from the cells transfected with vector containing IIS coding sequence. B, Im- munoblot analysis of IIS expression in COS-1 cells. Cell lysates from the culture of COS-1 cells expressing His-tagged IIS protein were resolved by SDS-PAGE under reducing (R) and nonreducing (NR) conditions, respectively, and then immunoblotted with anti-Xpress Ab. Lane 1, Lysate of cells transfected with vector only; lane 2, lysate of cells transfected with FIGURE 5. The induction of IIS mRNA expression in cultured lym- plasmid containing IIS cDNA encoding the mature polypeptide sequence 9 phoblast cells. A, The induction of IIS expression by cytokines (TNF-␣, without the leader peptide; lane 3, the lysate from cells transfected with IFN-␥, IL-4, and IL-13) in the indicated treatment time. B, The induction plasmid containing full-length IIS cDNA cloning sequence. C, Immunoblot of IIS expression by varying doses of IFN-␥. The levels of expression are analysis of IIS protein expression in lymphoblasts. Cell lysates from lym- represented as fold induction compared with the expression level in control phoblasts were immunoprecipitated with IIS Ab and resolved by SDS- cells. The experiments are repeated at least three times, and the results are PAGE under reducing conditions, and immunoblots were prepared using -Signif ,ء .expressed as the mean of fold induction Ϯ the SD of the mean IIS Ab. Note that the preadsorption of the antiserum with the synthetic icant differences (p Ͻ 0.05). oligopeptide, used as the Ag for generating IIS Ab, failed to recognize the IIS protein band. mRNA expression was markedly stimulated in activated CD8ϩ cells compared with that in resting cells (Fig. 6B). Most interestingly, activated CD19ϩ B cells expressed significantly higher lev- ␥ IIS is inducible by IFN- els of IIS mRNA compared with that in resting cells (Fig. 6B). Because it has been reported previously that the expression of UG is inducible by IFN-␥, we sought to determine whether the expres- Inhibition of IIS expression prevents lymphoblast cell migration sion of other members of this family of genes are also induced by and invasion IFN-␥. Accordingly, we determined the IIS mRNA levels by quan- Because it has been reported that IFN-␥-inducible protein-10 (IP- titative real-time PCR of total cellular RNA extracted from human 10) induces chemotaxis (31), we sought to determine whether IIS lymphoblast cells that were treated with IFN-␥ (10 ng/ml), TNF-␣ has any effect on cellular migration and invasion. Accordingly, (10 ng/ml), IL-4 (0.5 ng/ml), and IL-13 (10 ng/ml) for up to 6 h, lymphoblast cells were transfected with IIS antisense oligonucle- and compared the results with those from untreated controls. When otides and used for chemotaxis assay. Conditioned medium from lymphoblast cells were treated with IFN-␥ for 1 h, the expression fibroblast cell culture was used as the chemoattractant, and cell levels of IIS mRNA were significantly increased (Fig. 5A). How- migration and invasion assays were conducted as previously re- ever, the cells treated with cytokines other than IFN-␥ failed to ported (32, 33). The results show that chemotactic migration of show any stimulation of IIS mRNA expression (Fig. 5A). To de- antisense oligo-transfected cells was markedly inhibited compared termine a dose-related effect of IFN-␥ on the changes in IIS mRNA with that of the control and sense oligonucleotide-transfected cells expression, the cells were incubated with 0.1Ð10 ng/ml IFN-␥ for (Fig. 7A). Similarly, invasion of the antisense oligo-transfected 1 h. The expression levels of IIS mRNA were significantly in- cells was also significantly lower than that of control and sense creased in a dose-dependent manner up to 11.68-fold of nonstimu- oligonucleotide-transfected cells (Fig. 7B). These results suggest lated cells (Fig. 5B). This results show that IIS expression is in- that inhibition of IIS expression suppresses migration and invasion ducible by IFN-␥. in lymphoblast cells.

Stimulation of IIS expression in activated white blood cells Discussion To delineate which blood cells are speciﬁc for the expression of In this study, we characterized IIS. We assigned IIS to a novel IIS, we investigated its expression in different cell types of blood member of SCGB family based upon the following: 1) cDNA and cells by quantitative real-time PCR. IIS mRNA expression in var- amino acid sequences of IIS showed a high homology to those of ious types of resting WBCs was not signiﬁcantly different from SCGB family members, especially with LIP-B cDNA sequences that of lymphoblasts (Fig. 6A). IIS mRNA expression in CD4ϩ T showing a 72.6% homology; 2) IIS gene is mapped to human chro- cells was not induced in activated cells (Fig. 6B). However, IIS mosome 11, the region in which SCGB gene cluster is localized; 4250 IFN-␥-INDUCIBLE SCGB

FIGURE 6. The expression of IIS mRNA in different types of white blood cells. A, The expression level of resting cells from each cell type is FIGURE 7. The effect of IIS antisense S-oligonucleotide treatment of represented in fold increase compared with that of lymphoblasts. B,A lymphoblasts on cellular migration and invasion. The results of cell mi- comparison of the IIS expression levels in each type of activated WBCs gration assay are presented in A, and cell invasion assay in B. The results f u ( ) with that in the resting counterparts ( ). The results are expressed as are expressed as the total number of migrated or invaded cells per 0.005 ء Ϯ the mean of three independent experiments SD of the mean. , Signif- mm3. The results are expressed as the mean of three independent experi- Ͻ .(Signiﬁcant differences (p Ͻ 0.05 ,ء .icant differences (p 0.05). ments Ϯ SDs of the means

3) IIS gene structure is virtually identical with all genes in the and found to be associated with a rapid progression of IgA ne- SCGB family; and 4) IIS protein contains an N-terminal and a phropathy (39Ð41). Recently, it has been reported that UG gene central cysteine residue that are identical with those of LIP-A and polymorphisms affect the progression of IgA nephropathy by mod- -B, MGB-1 and -2, RYD-5, UGRP-1, and YGB, which are mem- ulating the level of expression of UG (40), which protects against bers of the SCGB family. abnormal renal glomerular deposition of IgA and fibronectin (42), The proteins in the SCGB family are divided into four groups characteristic of IgA nephropathy. Whether the SNP and STRP in according to the number of conserved cysteines. As indicated the IIS gene uncovered in the present study are predisposing fac- above, UG has two conserved cysteines at ϩ3 and ϩ69 positions tors for any human disease needs to be investigated. (Fig. 8A). Other members, such as LIP-A, LIP-B, MGB1, MGB2, The amino acid sequence deduced from IIS cDNA sequence and YGB (34), not only have the two cysteines at ϩ3 and ϩ69 but suggests that, like most SCGB family of proteins, this protein also also have an additional cysteine at ϩ44 position. In contrast, three contains a leader peptide. The founding member of this protein members of this family, RYD5, UGRP1, and UGRP2, have only family, UG, which is induced by IFN-␥, is also a secreted protein, one cysteine at the center of the molecule (35, 36). IIS is the only and recently, we have demonstrated that UG binds to as-yet-uni- member described so far that has two cysteines: one at the N- dentified cell surface binding protein(s) with high affinity and spec- terminal residue 3, and one at the center of the C terminus of the ificity and regulates cellular migration and invasion (32, 33, 43). molecule (residue 44). A comparison of the amino acid sequences Consistent with these findings, we find that IIS is also induced by between IIS and YGB shows that amino acid residues 1Ð77 in IFN-␥, and the suppression of IIS expression by antisense oligo- these two proteins are identical. However, the third cysteine resi- nucleotide treatment of the cells inhibits chemotactic migration due at position 78 of YGB is lacking in IIS, and from here the and invasion. It has been reported that an IFN-␥ response element amino acid residues are not identical. It is likely that a deletion of is present in the 5Ј promoter region of the UG gene (29). We also 1 nt in the YGB cDNA sequence may have occurred at codon ϩ78, detected a sequence in the promoter region of the IIS gene that is resulting in a frame shift that led to the origin of this new member. similar to the IFN-␥ response element in the UG promoter. An- The phylogenetic analysis also suggests that IIS and YGB may other IFN-␥-inducible protein, IP-10, which belongs to the super- have evolved from the same ancestral gene (Fig. 8B). family of chemokines, stimulates activation and recruitment of leu- We have detected both SNP and STRP in the IIS gene using kocytes as well as nonhemopoietic cells (44, 45). Furthermore, genomic DNA samples from 34 apparently normal subjects. Al- IFN-␥-inducible T cell ␣-chemoattractant has been reported to though the significance of these polymorphisms is unclear at this stimulate transendothelial migration of normal blood T lympho- time, the occurrence of a (G3A) SNP in the UG gene (19) has cytes (46). Interestingly, the deduced amino acid sequence of ma- been suggested to predispose individuals to asthma (19, 37, 38) ture IIS polypeptide bears 25% sequence identity with that of The Journal of Immunology 4251

FIGURE 8. Amino acid sequences of the human SCGB family of proteins and the SCGB phylogenetic tree. A, Alignment of amino acid sequences of SCGB family members. Accession numbers for the sequences were as follows: IIS, AY236538; UG, NM_003357; YGB (Ref. 34); LIP-A, NM_006552; LIP-B, NM_006551; MGB1, NM_002411; MGB2, NM_002407; RYD5*, BK 000201; UGRP2, NM_052863; UGRP1, NM_054023. Asterisk indicates that the accession number is for nucleotide sequences that were used for predicting the gene products. The consensus cysteines are represented in bold within boxes. B, Phylogenetic tree of the human SCGB family. This phylogenetic tree was generated after the alignment of the sequences using CLUSTAL W.

IP-10 for which receptor-mediated functions have been identified 7. Klug, J., H. M. Beier, A. Bernard, B. S. Chilton, T. P. Fleming, R. I. Lehrer, (reviewed in Ref. 47). It is tempting to speculate that IIS may also L. Miele, N. Pattabiraman, and G. Singh. 2000. Uteroglobin/Clara cell 10-kDa family of proteins: nomenclature committee report. Ann. NY Acad. Sci. 923:348. manifest similar properties as those of IP-10 and this SCGB, like 8. Mukherjee, A. B., G. C. Kundu, G. Mantile-Selvaggi, C.-J. Yuan, A. K. Mandal, IP-10, may have a receptor-mediated function. The facts that the S. Chattopadhyay, F. Zheng, N. Pattabiraman, and Z. Zhang. 1999. Uteroglobin: expression of this protein is augmented in activated CD8ϩ and a novel cytokine? Cell. Mol. Life Sci. 55:771. ϩ 9. Krishnan, R. S., and J. C. Daniel, Jr. 1967. “Blastokinin”: inducer and regulator CD19 cells, that its expression in lymphoblast cells is stimulated of blastocyst development in the rabbit uterus. Science 158:490. by IFN-␥, and that IIS antisense S-oligonucleotide treatment in- 10. Beier, H. 1968. Uteroglobin: a hormone-sensitive endometrial protein involved in hibits chemotactic migration and invasion, suggest that this protein blastocyst development. Biochim. Biophys. Acta 160:289. 11. Beato, M. 1976. Binding of steroids to uteroglobin. J. Steroid Biochem. 7:327. may have immunological functions. Future studies may delineate 12. Lopez de Haro, M. S., M. Perez Martinez, C. Garcia, and A. Nieto. 1994. Binding the molecular mechanism(s) by which IIS contributes to orches- of retinoids to uteroglobin. FEBS Lett. 349:249. trate the IFN-␥-mediated immune response. 13. Gillner, M., J. Lund, C. Cambillau, M. Alexandersson, U. Hurtig, A. Bergman, E. Klasson-Wehler, and J. A. Gustafsson. 1988. The binding of methylsulfonyl- polychloro-biphenyls to uteroglobin. J. Steroid Biochem. 31:27. Acknowledgments 14. Ray, M. K., S. Magdaleno, B. W. O’Malley, and F. J. DeMayo. 1993. Cloning We thank Dr. Krystyna Wisniewski for the generous gift of lymphoblast and characterization of the mouse Clara cell-specific 10-kDa protein gene: comparison of the 5Ј-flanking region with the human rat and rabbit gene. Biochem. cells used in this study. We also thank Drs. Ida Owens and Janice Y. Chou Biophys. Res. Commun. 197:163. for critical review of this manuscript and for helpful suggestions. 15. Gutierrez Sagal, R., and A. Nieto. 1998. Cloning and sequencing of the cDNA coding for pig pre-uteroglobin/Clara cell 10 kDa protein. Biochem. Mol. Biol. Int. References 45:205. 16. Dominguez, P. 1995. Cloning of a Syrian hamster cDNA related to sexual di- 1. Sen, G. C., and P. J. Lengyel. 1992. The interferon system: a bird’s eye view of morphism: establishment of a new family of proteins. FEBS Lett. 376:257. its biochemistry. J. Biol. Chem. 267:5017. 17. Muller-Schottle, F., A. Bogusz, J. Grotzinger, A. Herrler, C. A. Krusche, 2. Farrar, M. A., and R. D. Schreiber. 1993. The molecular cell biology of inter- K. Beier-Hellwig, and H. M. Beier. 2002. Full-length complementary DNA and feron-␥ and its receptor. Annu. Rev. Immunol. 11:571. the derived amino acid sequence of horse uteroglobin. Biol. Reprod. 66:1723. 3. Boehm, U., T. Klamp, M. Groot, and J. C. Howard. 1997. Cellular responses to interferon-␥. Annu. Rev. Immunol. 15:749. 18. Singh, G., S. L. Katyal, W. E. Brown, and A. L. Kennedy. 1988. Amino-acid and 4. Bach, E. A., M. Aguet, and R. D. Schreiber. 1997. The IFN receptor: a paradigm cDNA nucleotide sequences of human Clara cell 10 kDa protein. Biochim. Bio- for cytokine receptor signaling. Annu. Rev. Immunol. 15:563. phys. Acta 950:329. 5. Ehrt, S., D. Schnappinger, S. Bekiranov, J. Drenkow, S. Shi, T. R. Gringeras, 19. Zhang, Z., D. B. Zimonjic, N. C. Popescu, N. Wang, D. S. Gerhard, E. M. Stone, T. Gaasterland, G. Schoolnik, and C. Nathan. 2001. Reprogramming of the mac- N. C. Arbour, H. G. De Vries, H. Scheffer, J. Gerritsen, et al. 1997. Human rophage transcriptome in response to interferon-␥ and Mycobacterium tubercu- uteroglobin gene: structure, subchromosomal localization, and polymorphism. losis: signaling roles of nitric oxide synthase-2 and phagocyte oxidase. J. Exp. DNA Cell Biol. 16:73. Med. 194:1123. 20. Heyns, W., and P. DeMoor. 1977. Prostatic binding protein: a steroid-binding 6. Der, S. D., A. Zhou, B. R. Williams, and R. H. Silverman. 1998. Identification of protein secreted by rat prostate. Eur. J. Biochem. 78:221. genes differentially regulated by interferon ␣, ␤,or␥ using oligonucleotide ar- 21. Lea, O. A., P. Petrusz, and F. S. French. 1979. Prostatein: a major secretory rays. Proc. Natl. Acad. Sci. USA 95:15623. protein of the rat ventral prostate. J. Biol. Chem. 254:6196. 4252 IFN-␥-INDUCIBLE SCGB

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