IFN Regulatory Factor-1 Regulates IFN-γ -Dependent S Expression Karin Storm van's Gravesande, Matthew D. Layne, Qiang Ye, Louis Le, Rebecca M. Baron, Mark A. Perrella, Laura This information is current as Santambrogio, Eric S. Silverman and Richard J. Riese of September 26, 2021. J Immunol 2002; 168:4488-4494; ; doi: 10.4049/jimmunol.168.9.4488 http://www.jimmunol.org/content/168/9/4488 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. IFN Regulatory Factor-1 Regulates IFN-␥-Dependent Expression1

Karin Storm van’s Gravesande,* Matthew D. Layne,* Qiang Ye,* Louis Le,* Rebecca M. Baron,* Mark A. Perrella,* Laura Santambrogio,† Eric S. Silverman,* and Richard J. Riese2*

Cathepsin S is a cysteine with potent endoproteolytic activity and a broad pH profile. Cathepsin S activity is essential for complete processing of the MHC class II-associated invariant chain within B cells and dendritic cells, and may also be important in degradation in atherosclerosis and emphysema. Unique among cysteine , cathepsin S activity is up-regulated by IFN-␥. Given its importance, we sought to elucidate the pathway by which IFN-␥ increases cathepsin S expression. Our data demonstrate that the cathepsin S promoter contains an IFN-stimulated response element (ISRE) that is critical for IFN-␥- Downloaded from induced transcription in a cell line derived from type II alveolar epithelial (A549) cells. IFN response factor (IRF)-2 derived from A549 nuclear extracts associates with the ISRE oligonucleotide in gel shift assays, but is quickly replaced by IRF-1 following stimulation with IFN-␥. The time course of IRF-1/ISRE complex formation correlates with increased levels of IRF-1 and cathepsin S mRNA. Overexpression of IRF-1, but not IRF-2, markedly augments cathepsin S promoter activity in A549 cells. Furthermore, overexpression of IRF-1 increases endogenous cathepsin S mRNA levels in 293T epithelial cells. Finally, freshly isolated bone marrow cells from IRF-1؊/؊ mice fail to up-regulate cathepsin S activity in response to IFN-␥. Thus, IRF-1 is the critical transcriptional mediator of http://www.jimmunol.org/ IFN-␥-dependent cathepsin S activation. These data elucidate a new pathway by which IRF-1 may affect MHC class II processing and presentation. The Journal of Immunology, 2002, 168: 4488–4494.

athepsin S is a member of the -like family of cys- liberation of the Ii remnant from the class II-binding groove and teine proteases that reside within the endosomal compart- class II-peptide complex formation. The importance of cathepsin S ments and mediate proteolysis of endocytosed in regulating MHC class II-restricted Ag processing and presen- C Ϫ/Ϫ and polypeptides (1–3). Among this family, cathepsin S has unique tation is well illustrated by the phenotype of cathepsin S mice features pertinent to its biological activity. First, it exhibits a broad (6, 7). Inhibition of cathepsin S activity in B cells and dendritic pH profile and maintains a significant endoproteolytic activity at cells results in accumulation of class II-Ii complexes, attenuation by guest on September 26, 2021 neutral pH, suggesting that this may have important ex- of class II-peptide complex formation, and inability of these cells tracellular biological activity (1). Second, its constitutive expres- to present certain antigenic determinants. Dendritic cells derived sion is restricted primarily to bone marrow-derived APCs (4). from cathepsin SϪ/Ϫ mice have a marked defect of MHC class II Third, its mRNA expression, protein, and activity are up-regulated endosomal trafficking (9, 10). Finally, in human dendritic cells, by IFN-␥ (3) in several cell types (1, 2). proinflammatory cytokines give rise to a surge of MHC class II- Cathepsin S plays an essential role in regulation of MHC class peptide complex formation that is dependent on cathepsin S ac- II maturation and trafficking within APCs by mediating processing tivity (11). Thus, regulation of cathepsin S expression and activity 3 of class II-associated invariant chain (Ii) and exogenous Ags (4– has important consequences in control of MHC class II function 8). In B cells and dendritic cells, cathepsin S uniquely mediates the and subsequent CD4ϩ T cell stimulation. final cleavage of Ii to generate class II-associated invariant chain IFN-␥ is a potent regulator of cathepsin S expression in vascular peptide (CLIP). This cleavage is necessary to permit subsequent smooth muscle cells and in the lung parenchyma (12). Arterial smooth muscle cells stimulated with IFN-␥ exhibit enhanced ca- *Department of Medicine, Brigham and Women’s Hospital and Harvard Medical thepsin S activity and secretion, with a concomitant increase in School, Boston, MA 02115; and †Department of Cancer Immunology and AIDS, supernatant elastolytic activity, which is blocked with a selective Dana-Farber Cancer Institute, Boston, MA 02115 cathepsin S inhibitor (13). Furthermore, cathepsin S activity is in- Received for publication October 23, 2001. Accepted for publication February creased in walls of atheromas and aneurysms as compared with 28, 2002. normal arteries, suggesting that this enzyme may be playing a role The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance in breakdown of the extracellular matrix in atherosclerosis (14). with 18 U.S.C. Section 1734 solely to indicate this fact. Within the lung, transgenic mice generated to overexpress IFN-␥ 1 This work was supported by National Institutes of Health Grants 1UO1 HL65899 exhibit a chronic inflammatory cell infiltrate, increased cysteine and KO8 AI01555 (to R.J.R.). K.S.v.G. is the recipient of a grant from the Deutsche protease activity, and emphysematous-like changes in lung pathol- Forschungsgemeinschaft (STO 420/1-1). ogy and physiology (15, 16). Analysis of bronchoalveolar lavage 2 Address correspondence and reprint requests to Dr. Richard J. Riese, Division of Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital, Tower 4B, fluid of these transgenic mice shows that cathepsin S retains the 75 Francis Street, Boston, MA 02115. E-mail address: [email protected] most activity of the cysteine proteases in the extracellular envi- 3 Abbreviations used in this paper: Ii, invariant chain; CIITA, class II transactivator; ronment, consistent with its broad pH profile. Thus, IFN-␥-induced CLIP, class II-associated invariant chain peptide; 125I-JPM565, 125I-labeled JPM565; activation of cathepsin S has important physiological relevance in ICSBP, IFN consensus sequence-binding protein; IRF, IFN regulatory factor; ISGF3␥, IFN-stimulated gene factor 3␥; ISRE, IFN-stimulated response element; several disease processes, and elucidation of this pathway may ␤-gal, ␤-galactosidase. have direct clinical significance.

Copyright © 2002 by The American Association of Immunologists 0022-1767/02/$02.00 The Journal of Immunology 4489

IFN-␥ signaling involves ligand-induced oligomerization of labeling of cysteine proteases and IFN-␥ receptor subunits, leading to phosphorylation and activation immunoprecipitation of Janus kinase 1 and 2. This in turn leads to activation of the Cells were lysed in 50 ␮l 1% Triton X-100, 50 mM sodium acetate (pH dormant Stat1 molecule. Stat1 homodimers translocate to the nu- 4.2), and 1 mM EDTA on ice for 30 min. Postnuclear extracts were nor- cleus, where they direct transcription of specific target (17), malized to total protein content and incubated with 125I-labeled JPM565 including such secondary transcriptional activators as the family of (125I-JPM565; gift of H. Ploegh, Boston, MA) for 1 h at 37°C. Labeled IFN response factors (IRF), class II transactivator (CIITA) pro- lysates were then either subjected to SDS-PAGE right away (bone marrow ␤ cells), or immunoprecipitation, as described below (A549 cells). moters III and IV, and CCAAT/enhancing-binding protein- (18– Immunoprecipitation was performed, as described previously (21). Fol- 20). These transcriptional regulators are intermediaries in a com- lowing active site labeling of A549 cell lysates with 125I-JPM, SDS was plicated network that produces alterations in and added to bring the final concentration to 1%. Samples were boiled for 3 cellular function. min, neutralized, and diluted 10-fold with 0.5% Nonidet P-40, 50 mM Tris (pH 7.4), and 5 mM MgCl2. For the immunoprecipitation, samples were This study seeks to identify the molecular pathway by which precleared initially with 5 ␮l normal rabbit serum (Sigma-Aldrich) and 100 IFN-␥ induces up-regulation of the cathepsin S gene. We have ␮l protein A-agarose (Santa Cruz Biotechnology, Santa Cruz, CA), fol- identified a functional IFN-stimulated response element (ISRE) in lowed by a second preclear with 100 ␮l protein A-agarose alone. Immu- the cathepsin S promoter Ϫ100 bp from the transcriptional start noprecipitation was performed by incubating each sample with 10 ␮l anti- ␮ site. We show that, in human bronchial epithelial A549 cells, cathepsin S serum (gift of H. Chapman, San Francisco, CA) and 100 l ␥ protein A-agarose. Both preclears and immunoprecipitation steps were IRF-1 directly binds to this site and mediates IFN- -dependent conducted at 4°C for 1 h. Immunoprecipitated pellets were washed five transcriptional activation. These data elucidate a new pathway by times with 50 mM Tris (pH 7.4), 150 mM NaCl, and 3 mM EDTA. Pro- which IRF-1 may influence several important biological processes, teins were eluted from the beads by addition of SDS sample buffer. Sam- Downloaded from including maturation and trafficking of MHC class II molecules, ples were boiled for 5 min, analyzed by SDS-PAGE, and visualized by autoradiography. vascular remodeling, and elastin degradation within the lung.

Materials and Methods EMSA Cells and materials Nuclear extracts from A549 cells were prepared by a modification of the

method of Dignam et al. (22, 23) and described elsewhere. A double- http://www.jimmunol.org/ The type II alveolar epithelial (A549) cell line (American Type Culture stranded oligonucleotide for the IRSE consensus (Ϫ110 to Collection, Manassas, VA) and the 293T epithelial cell line were cultured Ϫ79, GATTTTAAATGAAACTGAAATGAAAGTT) was radiolabeled in RPMI 1640 supplemented with 10% heat-inactivated FBS (Life Tech- by filling in a 5Ј overhang with [␣-32P]deoxycytidine 5Ј-triphosphate and nologies, Rockville, MD), 10 mM glutamine, and penicillin/streptomycin. Klenow fragment (NEB, Beverly, MA) and purified by gel filtration Cells were maintained at 37°C and 5% CO2 in a humidified incubator and (Chroma Spin ϩST-10 columns; Clontech Laboratories, Palo Alto, CA). A passed at confluence every 4 days. mutated probe for the IRSE site was also generated (Ϫ110 to Ϫ79, Plasmids GATTTTAAATGAACTTAGAATGAAAGTT). Protein-DNA-binding reactions were performed with 5–10 ␮g nuclear A 892-bp fragment of the cathepsin S gene, using human genomic DNA as extract protein, 1 ␮l-labeled oligonucleotide (50,000 cpm), 1 ␮g poly(dI- a template, was amplified by the PCR, the product containing 859 bp of the dC), 1 ␮l salmon sperm DNA in 100 mM Tris (pH 7.5), 10 mM EDTA, 10 by guest on September 26, 2021 5Ј flanking region and the first 32 bp of exon 1. This fragment was sub- mM DTT, and 50% glycerol in a total volume of 20 ␮l. After incubation cloned into a pGL3-basic (Promega, Madison, WI) vector at the restriction at room temperature for 30 min, protein-DNA complexes were resolved on sites HindIII and KpnI and designated CatS (Ϫ859/ϩ32). To localize cis a 5% nondenaturing acrylamide gel in a 1ϫ Tris-borate-EDTA buffer at regulatory elements that may be important for IFN-␥ induction of cathepsin room temperature and visualized by autoradiography. S, we used CatS (Ϫ859/ϩ32) as a template to generate truncated cathepsin Supershift and cold competition experiments were performed by prein- S5Ј flanking regions by PCR. Constructs containing these 5Ј-deletion frag- cubating nuclear cell extracts with 2 ␮g specific polyclonal Abs (IRF-1, ments were designated CatS (Ϫ597/ϩ32), CatS (Ϫ375/ϩ32), CatS (Ϫ230/ IRF-2, IRF-4, IFN consensus sequence-binding protein (ICSBP), IFN- ϩ32), and CatS (Ϫ75/ϩ32). The full-length construct, CatS (Ϫ859/ϩ32), stimulated gene factor 3␥ (ISGF3␥), p50, p65; Santa Cruz Biotechnology) was also used as a template to mutate the downstream IRSE site (Ϫ100 to or 40 molar excess of annealed cold oligonucleotides, respectively, before Ϫ90, GAAACTGAAA to GAACTTAGAAT) by site-directed mutagenesis addition of labeled probes. (QuickChange XL site-directed mutagenesis kit; Stratagene, La Jolla, CA). DNA sequencing confirmed the accuracy of all constructs used in this Northern blot study. pSV-␤-galactosidase (␤-gal) plasmid containing the ␤-gal reporter gene was obtained from Promega. Plasmid DNA was purified from Esch- Total RNA from 1 ϫ 106 A549 or 293T cells was extracted using the erichia coli using the Qiagen plasmid purification system according to RNeasy mini kit (Qiagen). A quantity amounting to 15–20 ␮g total RNA manufacturer’s instruction (Chatsworth, CA). was fractionated by electrophoresis on a formaldehyde-agarose gel, trans- ferred to a nylon membrane, and probed with 32P-labeled cDNA probes. Transfection and preparation of cell extracts The cDNA probe for cathepsin S was generated by RT-PCR from total RNA extracted from A549 cells. The 345-bp cathepsin S cDNA probe, the Transient transfections were conducted in A549 cells, which were grown to ␤ 80% confluence in 60-mm dishes and transfected with LipofectAMINE rat GAPDH probe, the -actin probe, and IRF-1 probe were labeled with ␣ 32 Ј PLUS (Invitrogen, San Diego, CA) with 2 ␮g CatS constructs and 2 ␮g [ - P]deoxy cytidine 5 -triphosphate using the Prime-It II Random Primer pSV-␤-gal plasmid. Coexpression experiments were performed with labeling kit (Stratagene). The membrane was hybridized using QuickHyb solution (Stratagene) at 68°C for 60 min, washed according to manufac- IRF-1, IRF-2, and empty vector expression constructs. Each experiment Ϫ was conducted so that the sum of all plasmid concentrations transfected turer’s directions, and exposed to Kodak XAR film at 80°C. Equity of was equal. Transfected cells were stimulated with 50 ng/ml human rIFN-␥ sample loading was assessed by stripping and reprobing the membrane 32 ␤ (Sigma-Aldrich, St. Louis, MO) for 24 h, placed in lysis buffer (Promega), with P-labeled GAPDH or -actin cDNA. and subjected to one freeze-and-thaw cycle. Firefly luciferase activity was then assayed in lysates using the luciferase assay method (Promega). Lu- Western blot ciferase activity was normalized to ␤-gal activity to control for transfection efficiency. Nuclear extracts from unstimulated and stimulated A549 cells were used In some experiments (Fig. 6C), 293T cells were transfected with the and prepared, as described above. Protein concentrations were measured, IRF-1 expression construct using Fugene (Roche, Indianapolis, IN). Cells and equal amounts of nuclear extracts were subjected to a 15% SDS- were grown to 50–70% confluence in 8.5-cm tissue culture dishes, and PAGE, transferred to nitrocellulose membranes (Schleicher & Schuell, growth was arrested overnight in 8 ml serum-free RPMI medium. Trans- Keene, NH), and probed with either a specific IRF-1, IRF-2, or Sp3 Ab fection was performed by adding 18 ␮l Fugene reagent to 6 ␮g IRF-1 DNA (Santa Cruz Biotechnology). Membranes were incubated with secondary (or empty vector). Cells were incubated for 24 h, and Northern analysis HRP-conjugated anti-rabbit antiserum (Pierce, Rockford, IL), and detected was performed as described below. by chemiluminescence (Amersham, Arlington Heights, IL). 4490 IRF-1 REGULATION OF CATHEPSIN S EXPRESSION

Mice and tissue culture

tem1Mak Mice with a targeted mutation in the IRF-1 gene (B6.129-IRF1 ) and control mice (C57BL/6) were obtained from The Jackson Laboratory (Bar Harbor, ME). Male and female mice were used between 8 and 12 wk old. All animals were maintained under pathogen-free conditions at the animal facilities of Harvard Medical School (Boston, MA) in compliance with institutional guidelines. Isolation of bone marrow cells was performed, as previously described, following euthanasia with ketamine/xylazine (9). FIGURE 2. Cathepsin S mRNA is up-regulated by IFN-␥. Northern blot Results analysis showing cathepsin S mRNA expression in A549 cells. Cells were IFN-␥ up-regulates cathepsin S activity and mRNA expression in incubated with IFN-␥ (50 ng/ml) or medium alone for the indicated times. A549 cells As a control for even loading, the blots were stripped and reprobed for To determine whether cathepsin S activity is up-regulated in A549 GAPDH. cells, cells were stimulated with 50 ng/ml IFN-␥ for 4, 12, 24, and 48 h, and lysates were labeled with the suicide inhibitor 125I-JPM565 (21, 24). Subsequent to active site labeling, To further characterize the functional importance of the IRSE cathepsin S was immunoprecipitated (Fig. 1). In the absence of binding site within the cathepsin S promoter (Ϫ100 to Ϫ90), we IFN-␥, no significant cathepsin S activity was evident. Following mutated the consensus sequence site GAAACTGAAA to site ␥ stimulation with IFN- , there was a marked increase in cathepsin GAACTTAGAA. Three independent constructs were verified by Downloaded from S activity at 4 h. The activity levels of this enzyme continued to DNA sequencing. Although this mutation did not change basal increase for at least 48 h. Thus, cathepsin S activity is increased in activity (Fig. 3), it abolished the IFN-␥-dependent activation of the response to IFN-␥ in A549 cells. cathepsin S promoter (Fig. 3). Therefore, the ISRE consensus We next determined whether IFN-␥ also induced expression of binding site within the cathepsin S promoter is required for up- cathepsin S mRNA (Fig. 2). A549 cells were growth arrested in regulation by IFN-␥. serum-free medium for 24 h and subsequently treated with IFN-␥ http://www.jimmunol.org/ for 1, 2, 3, 4, 12, and 24 h. Expression of cathepsin S mRNA was IRF-1 binds to the IRSE in an IFN-␥-dependent manner monitored by Northern blotting. An increase in mRNA levels was Several members of the IRF family can bind to ISRE and influence observed after 1 h of IFN-␥ stimulation, with further increases at transcriptional regulation (25–27). To determine which IRF family 2, 3, 4, and 12 h (Fig. 2). The maximum induction was seen at 12 h member binds to the cathepsin S ISRE and activates this gene in with an 18-fold increase, as determined by densitometric analysis. A549 cells, we performed gel supershift analyses. These experi- Interestingly, cathepsin S mRNA levels decreased at 24 h of IFN-␥ ments were conducted using the cathepsin S ISRE oligonucleotide stimulation, whereas the protein activity level was maintained for sequence and Abs specific for IRF-1, IRF-2, IRF-4, ICSBP/IRF-8, at least 48 h (Fig. 1). and ISGF3␥ (p48, IRF-9) in A549 cells stimulated with IFN-␥ for

24 h (Fig. 4A). Abs against NF-␬B (p50, p65) were also used, by guest on September 26, 2021 ISRE consensus binding site is critical for IFN-␥ up-regulation because this transcription factor can form complexes with several in A549 cells IRF family members. In nuclear extracts derived from IFN-␥-stim- To localize the most important IFN-␥ response elements found in ulated A549 cells, IRF-1 binds directly to the ISRE sequence, as the cathepsin S promoter, we designed a series of promoter-re- porter deletion constructs. A deletion series was made with pro- gressively smaller fragments of the 5Ј region of the cathepsin S gene, cloned in tandem with the luciferase reporter gene. Con- structs were then transiently transfected into A549 cells for deter- mination of transcriptional activity and inducibility following stimulation with IFN-␥ (Fig. 3). IFN-␥ induction of the full-length CatS (Ϫ859/ϩ32) construct resulted in a 36-fold increase in lu- ciferase activity. Further deletions of the 5Ј region moderately de- creased the inducibility between 12- and 20-fold, but did not alter basal activity significantly. After the deletion of the IRSE consen- sus binding site (CatS (Ϫ75/ϩ32)), a marked loss of inducibility was observed, suggesting that the IRSE site is critical for IFN-␥ induction in the cathepsin S promoter.

FIGURE 3. ISRE is critical for IFN-␥-induced activation of the cathep- FIGURE 1. Cathepsin S activity is up-regulated by IFN-␥. A549 cells sin S promoter. A549 cells transiently transfected with deletion series con- were incubated for the indicated time with medium alone or IFN-␥ (50 structs or an ISRE-mutated construct were incubated for 24 h with medium ng/ml). Cell extracts were then solubilized and normalized to protein con- alone (Ⅺ) or 50 ng/ml IFN-␥ (f) before luciferase activity assay. Data tent. Lysates were then incubated with the cysteine protease active site represent fold induction based on the basal activity of the full-length con- inhibitor 125I-JPM565 to label the cysteine proteases, and subjected to im- struct (CatS) from three experiments (ϮSD). All transfections were nor- munoprecipitation with a specific Ab against cathepsin S. malized to ␤-gal activity. The Journal of Immunology 4491 Downloaded from

FIGURE 4. IRF-1 binds to IRSE following IFN-␥ stimulation in A549 cells. A, A549 cells were incubated for 24 h with 50 ng/ml IFN-␥. EMSAs were performed on nuclear extracts using a probe containing the IRSE site from the cathepsin S promoter. Supershifts were conducted using Abs against the indicated transcription factors. The supershifted band with the IRF-1 Ab is indicated by the arrow. The probe lane represents probe without nuclear extract.

The cold oligo lane represents binding competition with 40ϫ excess cold IRSE. The nonspecific oligo lane denotes binding competition with 40ϫ excess http://www.jimmunol.org/ 32P-labeled oligo with an unrelated sequence (Sp1). The mutant oligo lane represents binding of nuclear extracts with the mutated, 32P-labeled IRSE oligonucleotide. B, A549 cells were incubated with medium (Ϫ) or IFN-␥ (ϩ) for 24 h before EMSA supershift analysis. Analysis was performed as described in A above.

shown by a supershift of the IRF-1/ISRE complex following in- clear extracts for expression of IRF-1 and IRF-2 proteins prior to cubation with an Ab against IRF-1 (Fig. 4A). The other IRF family stimulation of these cells with IFN-␥ demonstrated no detectable members were not supershifted in IFN-␥-stimulated cells. This levels of IRF-1. Activation of these cells with IFN-␥ led to a rapid IRF-1 complex was specific, because a 40-fold molar excess of by guest on September 26, 2021 unlabeled identical oligonucleotide, but not unrelated oligonucle- otide, competed for IRF-1 binding and abolished the DNA-protein complex (Fig. 4A). Also, incubation of A549 nuclear extracts with a radiolabeled probe containing the mutated IRSE site resulted in complete loss of complex formation (Fig. 4A). To demonstrate further the specificity of IRF-1 in regulating IRSE-dependent gene expression, we compared the binding of IRF-1 and IRF-2 in A549 nuclear extracts derived from unstimu- lated and IFN-␥-stimulated cells (Fig. 4B). In unstimulated A549 cells, the Ab against IRF-2, but not IRF-1, supershifted the protein- DNA complex, implying that, in the absence of IFN-␥ stimulation, IRF-2 is able to associate with this oligonucleotide in vitro. How- ever, following activation with IFN-␥, the situation was reversed; the DNA-protein complex was supershifted with the IRF-1 Ab, indicating that IRF-1 had now replaced IRF-2. As shown in Figs. 1 and 2, the up-regulation of cathepsin S occurs relatively rapidly, with a noticeable increase in mRNA fol- lowing1hofstimulation. If indeed IRF-1 is the key element mediating increased transcription, one would expect IRF-1 binding to occur with a similar time course. A549 cells were stimulated FIGURE 5. IRF-1 expression and binding are rapidly up-regulated by with IFN-␥ for 1, 2, 3, 4, and 12 h, and nuclear extracts were IFN-␥. A549 cells were incubated for the indicated times with medium alone prepared (Fig. 5A). After1hofstimulation with IFN-␥, IRF-1 or with IFN-␥ before preparation of nuclear extracts. A, Time course of IRF-1 ␥ formed a complex with the IRSE oligonucleotide. This complex, binding to the ISRE probe following incubation of A549 cells with IFN- showing complex formation at 1 h. Specificity of binding reaction to the IRSE which persisted for at least 24 h, exhibited maximum protein-DNA ␥ consensus binding site was tested by supershift of band with IRF-1 Ab (arrow). complex formation at 2–3 h. Following IFN- stimulation, we B, Time course of IRF-1, IRF-2, and Sp3 protein expression in A549 cells could not supershift the DNA-protein complex with an Ab against following stimulation with IFN-␥ showing up-regulation of IRF-1, but not IRF-2 at any time point (data not shown). IRF-2, at 1 h. Cell lysates were obtained and normalized to protein content, The binding of IRF-1 to the ISRE in A549 cells could either followed by SDS-PAGE. Lysates were then transferred to nitrocellulose mem- result from activation (phosphorylation) of latent IRF-1 or from branes, and proteins were detected by immunoblot analysis with specific Abs. generation of new IRF-1 protein. Immunoblot analysis of the nu- SP3 was used to demonstrate equal loading of protein. 4492 IRF-1 REGULATION OF CATHEPSIN S EXPRESSION and robust increase in IRF-1 expression (Fig. 5B). The time course IRF-2 did not significantly inhibit IRF-1-inducible promoter ac- of IRF-1 expression correlated well with both the temporal in- tivity in cotransfection experiments (data not shown). Thus, IRF-1, crease in cathepsin S mRNA and the time course of IRF-1/IRSE but not IRF-2, is capable of inducing cathepsin S transcription, and complex formation. In contrast, IRF-2 expression did not change this gene activation is dependent on an intact IRSE site. appreciably following administration of IFN-␥. Thus, IFN-␥ exerts To determine whether IRF-1 can activate the endogenous ca- its IRF-1-mediated effects primarily through augmentation of thepsin S gene, Northern analysis of RNA from 293T epithelial IRF-1 expression. cells was performed following transfection of cells with empty expression vector (left lane), IRF-1 expression vector (middle IRF-1, but not IRF-2, increases cathepsin S promoter activity lane), or stimulation with 50 ng/ml IFN-␥ (right lane) (Fig. 6C). The above data show that the ISRE consensus site is required for The 293T cells were used for this experiment because the trans- IFN-␥-induced cathepsin S expression, and that IRF-1 derived fection efficiency in A549 cells (2–4%) was too low to detect from A549 nuclear extracts is capable of associating with this se- changes in cathepsin S expression by Northern analysis. The 293T quence. We wanted to examine whether IRF-1 expression could cells, like A549 cells, exhibit an IFN-␥-dependent increase in ca- increase cathepsin S promoter activity independent of IFN-␥.We thepsin S protein activity (data not shown). Cells were incubated cotransfected an IRF-1-containing expression vector into A549 for 24 h following transfection of IRF-1 before RNA extraction. cells and examined cathepsin S promoter activity. Transfection of Both transfection of IRF-1 and stimulation with IFN-␥ resulted in IRF-1 led to a dose-dependent increase in cathepsin S promoter a clear increase in cathepsin S mRNA in these cells (top panel). As activity as compared with transfection of empty vector (Fig. 6A). expected, IRF-1 transfection and IFN-␥ stimulation also resulted in an Mutation of the ISRE site resulted in a marked reduction of IRF- increase in IRF-1 mRNA levels (middle panel). Thus, IRF-1 is capa- Downloaded from 1-induced cathepsin S promoter activity, indicating that the IRF- ble of up-regulating the endogenous expression of cathepsin S. 1/ISRE complex formation at this site is necessary for promoter activity. In contrast, cotransfection of IRF-2 mildly repressed both IRF-1 mediates the IFN-␥-induced up-regulation of cathepsin S constitutive and inducible promoter activity (Fig. 6B). Titration of in mouse bone marrow cells

The functional significance of cathepsin S has been best elucidated http://www.jimmunol.org/ in professional APCs, in which it participates in Ii degradation and class II trafficking and maturation (4, 6, 7). In fact, the delivery of mature class II-peptide complexes to the cell surface of dendritic cells is associated with increased cathepsin S activity, and may even be physiologically regulated by manipulation of enzyme ac- tivity (11, 28). To investigate whether IRF-1 also regulates IFN- ␥-induced cathepsin S expression in APCs, bone marrow cells from wild-type and IRF-1Ϫ/Ϫ mice were isolated and incubated

overnight in the presence of IFN-␥ (50 ng/ml). Analysis of the by guest on September 26, 2021 murine cathepsin S promoter revealed a homologous IRSE con- sensus site at Ϫ26 bp upstream from the transcriptional start site. Bone marrow cells from IRF-1Ϫ/Ϫ mice displayed normal levels of constitutive cathepsin S expression (Fig. 7). This finding of normal constitutive cathepsin S expression also holds true for B cells, dendritic cells, and derived from IRF-1Ϫ/Ϫ animals (data not shown). However, stimulation of IRF-1Ϫ/Ϫ bone marrow cells with IFN-␥ failed to significantly increase activity levels of cathepsin S. A similar observation was reproduced in B cells de- rived from IRF-1Ϫ/Ϫ animals (data not shown). Thus, IRF-1 is critical for IFN-␥-induced cathepsin S activation in APCs, but does not regulate constitutive cathepsin S expression.

FIGURE 6. IRF-1, but not IRF-2, activates cathepsin S promoter. A, A549 cells were transiently cotransfected with the indicated reporter con- structs along with an empty control vector (0) or IRF-1 construct at 100 or 200 ng. Results are expressed as fold induction relative to the activity of each reporter in the absence of IRF-1 plasmid, and represent the mean Ϯ SD from three experiments. Luciferase activity was normalized to the ␤-gal activity for each sample. B, A549 cells were transiently cotransfected with the full-length cathepsin S promoter-reporter construct and with empty vector, IRF-1 (100 ng) or IRF-2 expression vectors (375 ng). Cells were incubated with medium alone or IFN-␥ for 24 h. Results are expressed as fold induction relative to the basal activity of the full-length construct. As FIGURE 7. IRF-1 is required for IFN-␥-induced cathepsin S up-regu- in A above, luciferase activity was normalized to ␤-gal activity for each lation in mouse bone marrow cells. Freshly isolated bone marrow cells sample. C, Growth-arrested 293T cells were transfected with empty vector from wild-type (WT) and IRF-1Ϫ/Ϫ mice were incubated with 50 ng/ml (left lane) or IRF-1 expression vector (middle lane), or stimulated with 50 IFN-␥ overnight. Lysates were obtained (6 ϫ 106 cells/sample), and cys- ng/ml IFN-␥ (right lane). Cells were incubated for 24 h before Northern teine proteases were labeled with 125I-JPM565. Samples were then ana- analysis for mRNA levels of cathepsin S (upper panel), IRF-1 (middle lyzed by SDS-PAGE, and cysteine protease activity was quantitated by panel), and ␤-actin (lower panel). A quantity amounting to 20 ␮g total autoradiography. The band corresponding to cathepsin S is indicated by the RNA was used for this blot. arrow. The Journal of Immunology 4493

Discussion dence that IRF-2 stimulates transcription of the cathepsin S gene. This is the first study investigating the transcriptional regulation of When present following stimulation with IFN-␥, IRF-1 may ex- cathepsin S, an enzyme that is critical for MHC class II processing hibit a higher affinity for the cathepsin S IRSE oligonucleotide than and presentation. Our data demonstrate that IFN-␥-induced acti- IRF-2, and thus IRF-2 binding is not detected in vitro (Figs. 5 and vation of cathepsin S is mediated by IRF-1 binding to a single 6). Overexpression of IRF-2 in the A549 cells moderately attenu- ␥ IRSE site 100 bp upstream from the transcriptional start site of the ates both the IFN- -induced and basal expression levels of cathep- cathepsin S promoter. The fact that IRF-1 is widely distributed sin S, although the degree of inhibition is lower than that observed among different cell types and tissues, and that APCs from IRF- for other genes (37). Ϫ Ϫ ␥ 1 / mice do not up-regulate cathepsin S expression in response to These data show that regulation of IFN- -stimulated cathepsin S IFN-␥, suggests that this pathway is more generally applicable to expression in A549 cells is largely controlled by IRF-1 binding to other cell types (26). The link between cathepsin S and IRF-1 has the ISRE in the cathepsin S promoter. Other members of the IRF ␥ several important implications. family, such as ICSBP, IRF-4, or ISGF3 (p48), did not associate Cathepsin S is highly expressed in alveolar macrophages and with this site. However, subtler pieces of evidence suggest that other bone marrow-derived APCs (1). Our data demonstrate that other factors may also influence transcriptional activation of this ϳ cathepsin S can also be expressed in A549 cells, which are derived protease. In the deletion series, we consistently observe an 2-fold from an epithelial alveolar type II cell, following induction with decrement in promoter activity with the first deletion construct Ϫ ϩ IFN-␥ (29). A549 cells are capable of expressing the necessary (CatS 596/ 32) as compared with the full-length construct (CatS Ϫ859/ϩ32). Thus, the ISRE site is necessary, but not suf- MHC class II and class II-related machinery to act as effective ficient for full activation of the cathepsin S promoter. In fact, this Downloaded from APCs when stimulated with IFN-␥, implicating a role for cathepsin region contains five CCAAT/enhancing-binding protein-␤ consen- S in processing of Ii and exogenous Ags for MHC class II-depen- sus elements and potential binding sites for high mobility group dent presentation in these cells (30). Furthermore, lung epithelial protein I/Y, which might facilitate the IFN-␥ response through cells, including type II epithelial cells, are potential sources for formation of higher order complexes (20). The exact pathway by production and secretion of elastinolytic proteases within the lung which these enhancer elements may augment IFN-␥-induced ca- (31). Consistent with this concept, selective overexpression of thepsin S transcription deserves further investigation. http://www.jimmunol.org/ IFN-␥ within the lung results in a production of chronic inflam- In summary, IFN-␥ stimulates cathepsin S expression via direct matory cell infiltrate and emphysema in a murine model of irre- association of IRF-1 with the cathepsin S promoter. Given the versible obstructive airway disease (15, 16). recent interest in cathepsin S regulation in several physiologic and IFN-␥ regulates both MHC class I and class II expression. How- pathologic processes, the elucidation of the transcriptional path- ever, the pathways by which IFN-␥ stimulates expression of these way controlling expression of this enzyme may give rise to novel proteins, and related molecules, are not identical. Similar to ca- therapeutic strategies. thepsin S, IRF-1 mediates the IFN-␥-stimulated MHC class II re- sponse, but does not appear to influence constitutive expression in professional APCs (32). Unlike cathepsin S, MHC class II expres- Acknowledgments by guest on September 26, 2021 sion is regulated by CIITA, the master transactivating regulatory We thank Dr. Jeffrey Drazen for his guidance and support, and Drs. Harold element for MHC class II, Ii, and HLA-DM (18, 19, 33). The Chapman, Hidde Ploegh, and Irvith Carvajal for their critical review of this cathepsin S promoter does not contain an X box consensus site, nor manuscript. does transfection of CIITA significantly alter cathepsin S expres- sion (data not shown). In contrast to MHC class II and cathepsin References S, both constitutive and IFN-␥-induced expression of MHC class 1. Shi, G. P., J. S. Munger, J. P. Meara, D. H. Rich, and H. A. Chapman. 1992. Ϫ/Ϫ Molecular cloning and expression of human alveolar cathepsin S, an I is markedly reduced in IRF-1 animals, presumably from at- elastinolytic cysteine protease. J. Biol. Chem. 267:7258. tenuated expression of TAP1 and low molecular mass polypeptide 2. Shi, G. P., A. C. Webb, K. E. Foster, J. H. Knoll, C. A. Lemere, J. S. Munger, 2. TAP1 and low molecular mass polypeptide 2 are regulated by and H. A. Chapman. 1994. Human cathepsin S: chromosomal localization, gene structure, and tissue distribution. J. Biol. Chem. 269:11530. IRF-1 binding directly to an IRSE element within their promoter 3. Chapman, H. A., R. J. Riese, and G. P. Shi. 1997. Emerging roles for cysteine segments (34). The transcriptional regulation of constitutive ca- proteases in human biology. Annu. Rev. Physiol. 59:63. thepsin S expression in APCs remains to be elucidated. 4. Riese, R. J., P. R. Wolf, D. Bromme, L. R. Natkin, J. A. Villadangos, H. L. Ploegh, and H. A. Chapman. 1996. Essential role for cathepsin S in MHC Initial studies on transcriptional regulation of the IFN-␣ and class II-associated invariant chain processing and peptide loading. Immunity IFN-␤ genes suggested that IRF-1 and IRF-2 act as transcriptional 4:357. activators and repressors of gene expression, respectively (35, 36). 5. Villadangos, J. A., R. J. Riese, C. Peters, H. A. Chapman, and H. L. Ploegh. 1997. Degradation of mouse invariant chain: roles of S and D and the in- This conclusion was based on studies showing that cotransfection fluence of major histocompatibility complex polymorphism. J. Exp. Med. 186: of IRF-1 increased expression of IFN-␣␤, whereas cotransfection 549. 6. Shi, G. P., J. A. Villadangos, G. Dranoff, C. Small, L. Gu, K. J. Haley, R. Riese, of IRF-2 reduced this IRF-1-mediated activation. A more recent H. L. Ploegh, and H. A. Chapman. 1999. Cathepsin S required for normal MHC study showed that IRF-1 mediated the activation of cyclooxygen- class II peptide loading and germinal center development. Immunity 10:197. ase 2 expression, whereas IRF-2 inhibited both the constitutive and 7. Nakagawa, T. Y., W. H. Brissette, P. D. Lira, R. J. Griffiths, N. Petrushova, J. Stock, J. D. McNeish, S. E. Eastman, E. D. Howard, S. R. Clarke, et al. 1999. IRF-1-induced activation of this gene (37). Macrophages from Impaired invariant chain degradation and and diminished Ϫ Ϫ IRF-2 / mice exhibited an increase in both basal and IFN-␥- -induced arthritis in cathepsin S null mice. Immunity 10:207. induced cylooxygenase 2 expression, consistent with the paradigm 8. Riese, R. J., R. N. Mitchell, J. A. Villadangos, G. P. Shi, J. T. Palmer, E. R. Karp, G. T. De Sanctis, H. L. Ploegh, and H. A. Chapman. 1998. 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