Intracellular IL-1�-binding proteins contributeto biological functions of endogenous IL-1�in systemic sclerosis fibroblastsYasushi Kawaguchi*, Emi Nishimagi, Akiko Tochimoto, Manabu Kawamoto, Yasuhiro Katsumata, Makoto Soejima,Tokiko Kanno, Naoyuki Kamatani, and Masako Hara

Institute of Rheumatology, Tokyo Women’s Medical University, 10-22 Kawada-cho, Shinjuku-ku, Tokyo 162-0054, Japan

Edited by Charles A. Dinarello, University of Colorado Health Sciences Center, Denver, CO, and approved August 8, 2006 (received for review May 2, 2006)

The aberrant production of precursor IL-1� (pre-IL-1�) in skinfibroblasts that are derived from systemic sclerosis (SSc) is associ-ated with the induction of IL-6 and procollagen, which contributesto the fibrosis of SSc. However, little is understood about howintracellular pre-IL-1� regulates the expression of the other mol-ecules in fibroblasts. We report here that pre-IL-1� can form acomplex with IL-1�-binding proteins that is translocated into thenuclei of fibroblasts. Immunoprecipitation that used anti-humanIL-1� Ab and 35S-labeled nuclear extracts of fibroblasts showedthree specific bands (�31, 35, and 65 kDa). The 31-kDa moleculewas identified as pre-IL-1�, and the 35- and 65-kDa moleculesmight be pre-IL-1�-binding proteins. A partial sequencing for the10 aa from the N-terminals of the molecules showed 100% homol-ogy for HAX-1 (HS1-associated protein X-1) and IL-1 receptor typeII (IL-1RII). Suppression of the genes of HAX-1 or IL-1RII induced theinhibitory effects of IL-1 signal transduction, including productionof IL-6 and procollagen, by fibroblasts. In particular, pre-IL-1� wasnot translocated into the nucleus by an inhibition of HAX-1. Thesefindings reveal that nuclear localization of pre-IL-1� depends onthe binding to HAX-1 and that biological activities might be elicitedby the binding to both HAX-1 and IL-1RII in SSc fibroblasts.

IL-1 receptor type II � HS1-associated protein X-1 � fibrosis � collagen � IL-6

Systemic sclerosis (SSc) is a connective tissue disease ofunknown etiology that is characterized by the fibrosis of

systemic organs (1). Because skin thickening manifests in mostpatients, researchers have analyzed the molecular and biologicalfunctions of lesional skin fibroblasts that are derived from SScpatients (2). In previous reports we demonstrated that SScfibroblasts expressed IL-1� mRNA constitutively and that ab-errant production of precursor IL-1� (pre-IL-1�) contributed toskin fibrosis in SSc (3–5). IL-1� is a multifunctional moleculethat is involved in a variety of inflammatory disorders, includingsepsis, arthritis, myositis, psoriasis, periodontitis, and Alzhei-mer’s disease (6). Pre-IL-1� is synthesized as a result of thetranscription and translation of the IL1A gene. Under somecircumstances, pre-IL-1� (31 kDa) is proteolytically cleaved toyield a mature form of IL-1� (17 kDa) (7). Because theN-terminal propiece of pre-IL-1� (NTP-IL-1�) contains a nu-clear localization sequence (NLS), pre-IL-1� can be translocatedinto the nucleus, whereas mature IL-1� can be released fromcells (8).

This pathway is complicated, however. The signal transductionof IL-1� is initiated by the binding of IL-1� (precursor or matureform) to cell-surface receptors on various cells (IL-1 receptortype I and IL-1 receptor accessory protein) (9, 10). The intra-cellular accumulation of pre-IL-1� in skin fibroblasts suggests analternative pathway. Only a few studies, including ours, havereported the biological effects of intracellular IL-1� in fibro-blasts and endothelial cells (11–15). Although the precise path-way of signal transduction was not determined in those studies,the authors speculated that intracellular pre-IL-1� might exhibit

a biological function directly and that the pathway of signaltransduction might be distinct from the pathway that was me-diated by binding the specific receptors. Our previous study (5)revealed that intracellular pre-IL-1� directly influenced thephenotype of SSc fibroblasts. These observations prompted us toexplore the mechanism whereby intracellular pre-IL-1� exhibitsits biological functions through the alternative pathway. In thepresent study we investigate the molecules that bind to pre-IL-1�in human fibroblasts and the effects of the IL-1�-bindingproteins on nuclear localization and biological functions ofIL-1�.

ResultsLocalization of Intracellular IL-1� in SSc Fibroblasts. Although wepreviously demonstrated the nuclear localization of pre-IL-1� inSSc fibroblasts, we performed immunohistochemistry on fivelines of SSc fibroblasts and three lines of normal fibroblasts. Wevisualized the signals of intracellular IL-1� in all five SScfibroblast lines and did not detect them in the three normalfibroblast lines. A representative result of Cy3 staining is shownin Fig. 1. The specific signals were mostly distributed in thenucleus, consistent with our previous results (5).

Immunoprecipitation (IP). To detect candidates of intracellularIL-1�-binding proteins, we used cell lysates of SSc fibroblastsand anti-IL-1� Ab to perform IP. As shown in Fig. 2, autora-diography indicated that the lengths of the specific bands were�31, 35, and 65 kDa. Columns 2 and 3 show representative datafrom IP that use cell lysates of SSc fibroblasts with anti-IL-1� Abunder a nonreducing and a reducing condition, respectively, andcolumn 1 shows data that use cell lysates of SSc fibroblasts withrabbit IgG under a reducing condition. The 31-kDa band cor-responded to the predicted pre-IL-1� in fibroblasts. Because the35- and 65-kDa bands were also candidates of the intracellularIL-1�-binding proteins, we used a protein sequencer to partiallyanalyze the N-terminals of these molecules. The 35-kDa mole-cule was homologous to HAX-1 (HS1-associated protein X-1;amino acid sequence MSLFDLFRGF), and the 65-kDa mole-cule was homologous to IL-1 receptor type II (IL-1RII; aminoacid sequence FTLQPAAHTG). We observed no specific bandsbelow the 30-kDa molecule (data not shown). Thus, we con-

Author contributions: Y. Kawaguchi, E.N., A.T., Y. Katsumata, M.S., T.K., N.K., and M.H.designed research; Y. Kawaguchi, E.N., A.T., and M.K. performed research; Y. Kawaguchi,E.N., A.T., M.K., Y. Katsumata, M.S., T.K., N.K., and M.H. analyzed data; and Y. Kawaguchiwrote the paper.

The authors declare no conflict of interest.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: Ct, threshold cycle; HAX-1, HS1-associated protein X-1; icIL-1RA, intracellu-lar IL-1 receptor antagonist; IL-1RII, IL-1 receptor type II; IP, immunoprecipitation; NTP-IL-1�, N-terminal propiece of pre-IL-1�; pre-IL-1�, precursor IL-1�; SSc, systemic sclerosis.

*To whom correspondence should be addressed. E-mail: y-kawa@ior.twmu.ac.jp.

© 2006 by The National Academy of Sciences of the USA

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cluded that intracellular IL-1� was almost pre-IL-1� (31 kDa) inSSc fibroblasts, consistent with our previous studies (3–5).

Expression of IL-1RII and HAX-1 in Fibroblasts. To confirm theexpression of IL-1RII and HAX-1 in SSc and normal fibroblasts,we used RT-PCR to analyze the expression of mRNA. ThecDNA that was derived from five fibroblast lines of patients withSSc contained mRNA of both IL-1RII and HAX-1, but threenormal fibroblast lines contained the HAX-1 mRNA alone(Table 1). Western blotting indicated that HAX-1 was expressedin SSc and normal fibroblasts, but we detected IL-1RII in SScfibroblasts alone (Fig. 3). Immunocytochemical studies revealeddifferent distributions of HAX-1 and IL-1RII between SSc andnormal fibroblasts (Fig. 4). HAX-1 was localized in the nucleiand cytosol of SSc fibroblasts but in only the cytosol of normalfibroblasts. IL-1RII was localized in the nuclei and cytosol of

SSc fibroblasts, and no fluorescent signal was detected in normalfibroblasts.

Binding Capacities of IL-1RII to Pre-IL-1� in Fibroblasts. To investi-gate the binding capacities of IL-1RII to pre-IL-1� in fibroblasts,we produced murine fibroblasts that were transfected withhuman pre-IL-1�, human IL-1�, or human NTP-IL-1�, whichwere cotransfected with human IL-1RII. We detected humanIL-1RII, which was expressed by the pcDNA3 vector, in murinefibroblasts by anti-IL-1RII Ab (Fig. 5, second row). Because thethree forms of human IL-1� were expressed as V5-taggedproteins in murine fibroblasts, these proteins were detected byanti-V5 Ab (Fig. 5, third row). Finally, after cell lysates frommurine fibroblasts were immunoprecipitated with anti-IL-RIIAb, human IL-1� and human pre-IL-1� were detected byWestern blotting with anti-V5 Ab (Fig. 5). These results indicatethat intracellular IL-1RII binds to pre-IL-1� and mature IL-1�via the amino acid sequence from 113 to 271 aa.

To confirm the interaction of IL-1� with IL-1RII and HAX-1,we fused cDNA that encodes three kinds of IL-1� to the �repressor protein (�cI) of pBT plasmid and fused each targetgene of IL-1RII and HAX-1 to the N-terminal domain of RNApolymerase of pTRG plasmid. We grew double transformingcells of pre-IL-1� with IL-1RII or HAX-1 in selection LB platesand had a �-galactosidase activity (Fig. 6). The cells transformedwith NTP-IL-1� were positive only when cotransformed withHAX-1, and the cells transformed with IL-1� were positive onlywhen cotransformed with IL-1RII. Previously, HAX-1 wasreported to be associated with three sites of NTP-IL-1� (16). Byconsidering all this evidence, we present a schematic of putativepre-IL-1� complex in Fig. 7.

Functional Roles of IL-1RII and HAX-1 in the Signal Transduction ofPre-IL-1�. To further investigate the roles of IL-1RII and HAX-1as a pre-IL-1�-binding protein, we produced SSc fibroblasts thatdeplete IL-1RII or HAX-1 by means of RNA interference.

Fig. 1. Nuclear localization of IL-1� in SSc fibroblasts. SSc fibroblasts werecultured in plates on a four-chamber slide. Cells were fixed with 2% parafor-maldehyde plus 0.1% Triton X-100. The primary Ab was monoclonal anti-human IL-1� Ab, which detected pre-IL-1� and mature IL-1� after incubationwith Cy3-conjugated anti-mouse IgG Ab. A representative result in SSc fibro-blasts was obtained with fluorescence microscopy.

Fig. 2. Pre-IL-1�-binding proteins were detected by IP. Fibroblasts from SScwere cultured by using [35S]methionine�cystein for 16 h. After a pulse, cellswere harvested and sonicated to extract nuclear and cytosolic proteins. IP wasperformed with cell lysates and anti-human IL-1� Ab or control rabbit IgGcombined with protein G-Sepharose. Immunoprecipitates were fractionatedby 10% SDS�PAGE, and radiolabeled polypeptides were visualized by auto-radiography. Column 1, lysate reacted with rabbit IgG fractionated under areducing condition; column 2, lysate reacted with anti IL-1� Ab fractionatedunder a nonreducing condition; column 3, lysate reacted with anti IL-1� Abfractionated under a reducing condition; column M, molecular marker.

Fig. 3. Western blot analysis of IL-1RII and HAX-1 in fibroblasts. Cell lysateswere prepared from fibroblasts derived from systemic sclerosis (SSc) and ahealthy donor (HC).

Table 1. Expression of IL-1RII and HAX-1 mRNA in culturedfibroblasts

Subjects IL-1RII HAX-1

SSc1 17 4.32 15 5.23 29 424 31 265 25 18

HC1 �0.01 202 0.02 213 �0.01 48

Total RNA was extracted from cultured fibroblasts derived from five pa-tients with SSc and three healthy controls (HC). Real-time RT-PCR was per-formed by using an ABI 7900HT and FAM-labeled TaqMan gene expressionassay kit. GAPDH mRNA expression was used as an endogenous control.

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IL-1RII and HAX-1 proteins were suppressed in all five lines ofSSc fibroblasts transfected with a small siRNA-expressing vec-tor. A representative result of Western blotting is shown in Fig.8. We used five lines of SSc fibroblasts to conduct the experi-ments, and then we scanned each band on x-ray films on ascanning densitometer. We measured the intensity of eachmolecule by subtracting the intensity of background from that ofthe band. The mean ratio of specific�random siRNA was 0.06 inIL-1RII and 0.21 in HAX-1. An inhibition of IL-1RII did notaffect the nuclear localization of pre-IL-1�, but inhibitingHAX-1 caused the nuclear staining of pre-IL-1� in SSc fibro-blasts to disappear (Fig. 9). We used five different lines of SScfibroblasts to confirm this result. We previously demonstratedthat aberrant production of pre-IL-1� in the nucleus contributedto IL-6 and procollagen type I production in SSc fibroblasts. Toexplore the effects of IL-1RII and HAX-1 on IL-6 and procol-lagen type I production in SSc fibroblasts, we suppressed theproduction of both IL-6 and procollagen type I by the knock-down of IL-1RII and HAX-1 (Fig. 10 A and B). The resultsindicate the mean of triplicate experiments that use five SScfibroblasts and three normal fibroblasts.

DiscussionThe results of the present study provide solid evidence thatintracellular pre-IL-1� consists of a protein complex with IL-1RII and HAX-1 and that the formation of this complex isindispensable for pre-IL-1�-induced biological functions (IL-6production and procollagen type I synthesis by fibroblasts). Ourprevious findings indicated that nuclear localization of pre-IL-1�plays a crucial role in the fibrogenic phenotype of skin fibroblastsderived from patients with SSc (3–5). The present study dem-onstrates the importance of the pre-IL-1� complex for thefibrogenic phenotype of SSc fibroblasts.

Early research indicated that IL-1� or pre-IL-1� is secretedfrom cells and exhibited an inflammatory response and immu-nity through the specific IL-1 receptors on the surface oftargeted cells. However, intracellular pre-IL-1� has been shownto stimulate proliferation of renal fibroblasts (11) and to regulatethe migration and the life span of endothelial cells (12), inde-pendent of secretion and cell-surface IL-1 receptors. Someresearchers suggested that the nuclear localization sequence inthe NTP-IL-1� molecule might be essential for the biologicalactivity of intracellular IL-1� (11, 12). Recently, Buryskova et al.(13) observed that intracellular pre-IL-1� functionally activatedtranscription, interacting with histone acetyltransferase com-plexes. Werman et al. (14) reported that intracellular IL-1� is

Fig. 4. Cellular distribution of IL-1RII and HAX-1 by immunofluorescencestaining. Fibroblasts were fixed by 2% paraformaldehyde plus 0.1% TritonX-100 and then reacted with anti-IL-1RII or anti-HAX-1 Ab. After they weretreated with FITC-conjugated anti-mouse IgG, a fluorescence image wasobtained.

Fig. 5. A binding assay of pre-IL-1� and IL-1RII was performed with murinefibroblasts (NIH 3T3) transfected with human IL-1� and IL-1RII. The cDNA ofhuman IL-1� (amino acids 113–271), pre-IL-1� (amino acids 1–271), and NTP-IL-1� (amino acids 1–112) were subcloned into the pcDNA4-V5 vector. ThecDNA of human IL-1RII was subcloned into the pcDNA3 vector. NIH 3T3 cellswere transfected with one of three kinds of pcDNA4-V5 and pcDNA3, asindicated above the panels. Cell lysates were extracted from each transfectantby sonication. The upper three panels indicate the results of Western blotting(WB) with anti-IL-1� Ab, anti-IL-1RII Ab, and anti-V5 Ab. The lowest panelindicates the results of WB with anti-V5 Ab after IP that used anti-IL-1RII Ab.

Fig. 6. A bacterial two-hybrid system was performed to confirm the inter-action between pre-IL-1� and its binding proteins (IL-1RII and HAX-1). TheIL-1� proteins were fused to the bacteriophage � repressor protein by usingpBT plasmid, and the target proteins (IL-1RII and HAX-1) were fused to theN-terminal domain of RNA polymerase by using pTRG plasmid. The suitable E.coli was transformed by the two plasmids, and LB agar plates, includingtetracycline, chloramphenicol, kanamycin, and X-gal, were used to selectpositive clones.

Fig. 7. A putative structural component of the pre-IL-1� complex in fibro-blasts. IL-1RII binds to the C-terminal domain of pre-IL-1�, and HAX-1 binds tothe N-terminal domain in fibroblasts. However, unknown proteins, aside fromthese two, may bind to pre-IL-1� and unknown proteins may directly bind toIL-1RII or HAX-1. The component proteins within the pre-IL-1� complex maypossess the DNA- or RNA-binding motif, which may allow the pre-IL-1�

complex to modulate the fibrosis and inflammation.

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involved in the transcriptional activation of several proteins.Although IL-1� was traditionally understood to exhibit biolog-ical functions such as inflammation, autoimmunity, and fibrosisthrough IL-1 receptors on the cell surface, the abovementionedfindings and this study strongly support the theory of a nuclearsite of action for IL-1�.

Another important finding is a role of IL-1RII, which bindspre-IL-1� inside human fibroblasts that are derived from SSc.McMahon et al. (17) and Sims et al. (18) reported that IL-1RIIis a cell-surface receptor on B lymphocytes and neutrophils witha binding affinity for IL-1�, pre-IL-1�, and IL-1�, but it is notcapable of the signal transduction of IL-1 because of the lack ofthe endoplasmic domain. Our current results revealed thatIL-1RII combined with pre-IL-1� plays a crucial role in thebiological features of pre-IL-1� within SSc fibroblasts. We alsofound the differential expression of IL-1RII between SSc (n �5) and normal fibroblasts (n � 3) at the cellular mRNA andprotein levels. Constitutive expression of IL-1RII, as well aspre-IL-1�, may be an important phenotype of SSc fibroblasts,although the mechanisms whereby intracellular IL-1RII washighly expressed in SSc fibroblasts remain to be clarified.

Suzuki et al. (19) first identified the HAX-1 protein byscreening the proteins that interact with HS1 (hematopoieticlineage cell-specific protein 1). HS1 is B cell-signaling proteinand is one of the major substrates of the Src and Syk�Zap-70kinases (20). The HS1 protein mainly exists in the cytoplasm and

nucleus, and, when the molecule is associated with HAX-1, itmoves to the mitochondrial membrane. HAX-1 also interactswith pre-IL-1� in human chondrocytes, although the biologicalproperties for the complex of HAX-1 and pre-IL-1� have notbeen fully elucidated (16). The HAX-1 protein appears to beexpressed ubiquitously in various normal tissues and to consti-tute the domain that is responsible for binding to the pre-IL-1�,HS1, cortactin, PKD2, EBNA-LP, Bcl-2, and HIV1 Vpr proteins(21–23). The fact that HAX-1 interacts with a variety of struc-turally unrelated proteins suggests an essential function forHAX-1 that involves intracellular signaling and shuttling ofvarious intracellular molecules. Our observations indicate theimportance of HAX-1 for the nuclear localization of pre-IL-1�in fibroblasts. Posttranslational modifications such as phosphor-ylation and myristoylation of NTP-IL-1� are well recognizedmechanisms that are involved in the transport of pre-IL-1� to thenucleus (24, 25). Notably, myristoylation occurs on lysine resi-dues 82 and 83 of pre-IL-1�, located in the nuclear localizationsequence (NLS). HAX-1 was associated with three segments ofNTP-IL-1�, including the NLS segment (16), which suggests thatthe binding of HAX-1 with the NLS (KVLKKRR) of pre-IL-1�might facilitate the nuclear localization of the pre-IL-1� complexin fibroblasts. Taken together, the findings strengthen the con-clusion that proteins associated with HAX-1 can shuttle betweennuclear and cytoplasmic compartments.

A previous investigation looking for a nuclear target ofpre-IL-1� revealed the interaction between pre-IL-1� and nec-din by a yeast two-hybrid system (26). Necdin is a 47-kDa proteinthat functions as a cell-growth suppressor in a manner similar tothat of the retinoblastoma tumor suppressor protein, Rb (27, 28).In our study, IP showed a faint band (�47 kDa) that wassubjected to N-terminal amino acid sequence analysis. However,we could not identify the molecule because of the small amountof peptide. Although we did not confirm that necdin was one ofthe intracellular pre-IL-1�-binding proteins, we did detect theexpression of necdin in SSc and normal fibroblasts (data notshown). Moreover, the suppression of necdin with an RNAimethod did not affect IL-6 and procollagen type I production inSSc fibroblasts (data not shown), which is inconsistent with theresults of previous studies. This discrepancy may be explained,in part, by the different cell types used in each experiment(fibroblasts versus Saos-2 osteosarcoma cells).

Fig. 8. Depletion of IL-1RII and HAX-1 by RNA interference. A DNA fragmentthat targeted the sequence in the ORFs of IL-1RII and HAX-1, and a controlwith a corresponding random sequence were obtained and were cloned intopSilencer 3.1H1-neo, an siRNA-expressing vector. Stable transfectants selectedby G418 were cultured in DMEM plus 10% FBS for 72 h, and cell lysates wereprepared by sonication. The cell lysates from various transfectants wereresolved in 15% SDS�PAGE and transferred to nitrocellulose membrane.Anti-IL-1RII Ab (Upper) and anti-HAX-1 Ab (Lower) were used to performWestern blotting (WB). Lane 1, siRNA vector that contained the sequences ofIL-1RII; lane 2, siRNA vector that contained random sequences for IL-1RII; lane3, siRNA vector that contained the sequences of HAX-1; lane 4, siRNA vectorthat contained random sequences for HAX-1.

Fig. 9. Cell distribution of pre-IL-1� in SSc fibroblasts depleting IL-1RII orHAX-1. We obtained a DNA fragment targeting the sequence of IL-1RII orHAX-1, which was cloned into pSilencer 3.1 H1-neo, an siRNA-expressingvector. As a control, a scramble DNA fragment was generated that had thesame number of nucleotides but did not display sequence identity with IL-1RIIand HAX-1 (random siRNA). These vectors were transfected into SSc fibro-blasts, and intracellular pre-IL-1� was detected by using immunocytochemis-try of DAB staining.

Fig. 10. IL-6 and procollagen type I C-peptide production decreases in SScfibroblasts by the suppression of IL-1RII or HAX-1. Fibroblasts were cultured inserum-free media. After 48 h of culturing, commercial ELISA kits were used tomeasure IL-6 (A) and procollagen type I C-peptide (B) in culture supernatants.Open bars, random siRNA transfectants; filled bars, specific siRNA transfec-tants; SSc, fibroblasts derived from SSc (n � 5); HC, fibroblasts derived fromhealthy controls (n � 3). *, P � 0.05; **, P � 0.01 (compared with random siRNAtransfectants).

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Recent reports by Higgins et al. (29) and Kanangat et al. (30)demonstrated the biological functions of intracellular IL-1 re-ceptor antagonist (icIL-1RA) in SSc fibroblasts. They indicatedthat icIL-1RA was overexpressed in SSc fibroblasts and thaticIL-1RA was involved in the fibrogenic phenotype of SScfibroblasts. Although we did not examine the expression oficIL-1RA in this study, icIL-1RA may have bound to intracel-lular IL-1RII that consisted of the pre-IL-1� complex. Todetermine whether icIL-1RA is the fourth component of thepre-IL-1� complex in SSc fibroblasts would have a potential rolein delineating the molecular events of the fibrosis in SSc that areassociated with the pre-IL-1� complex.

In conclusion, our study found the formation of the pre-IL-1�complex, which consists of pre-IL-1�, IL-1RII, and HAX-1,inside SSc fibroblasts. This complex plays a crucial role in thefibrogenic phenotype of SSc fibroblasts. Because of its nuclearlocalization, we believe this complex acts in the nuclei offibroblasts; however, based on a search of the National Centerfor Biotechnology Information conserved domain database,these proteins do not have a DNA-binding motif. We speculatethat this complex is part of a larger one. A putative pre-IL-1�complex is illustrated in Fig. 7.

Materials and MethodsCell Culture. After providing informed consent, five female pa-tients with SSc (median age 46) and three healthy female donors(median age 42) were enrolled in this study, which met thestandards of our institutional review board. All patients wereclassified into diffuse cutaneous SSc according to the criteria ofthe American Rheumatism Association (31) and the classifica-tion of LeRoy et al. (32). Skin fibroblast lines were obtained frombiopsied skin and explanted into tissue cultures. A murinefibroblast-like cell line, NIH 3T3, was also used in this study andwas obtained from the American Tissue Culture Collection. Theculture media consisted of DMEM (Sigma, St. Louis, MO) with10% FBS (Sigma) and antibiotics (penicillin and streptomycin;Invitrogen, Carlsbad, CA) or of a serum-free medium (QBSF-51; Sigma). In this experiment, cells were used in the thirdthrough the fifth passages.

Immunocytochemical Staining. Monolayer fibroblast cultures (5 �103 cells per well) were grown for 48 h in four-chamber slides(Lab-Tek; Nalge Nunc, Tokyo, Japan). Fibroblasts were washedtwice with cold PBS and fixed with 2% paraformaldehyde plus0.1% Triton X-100 in PBS. The primary Abs used in thisexperiment were monoclonal anti-human IL-1� Ab (R & DSystems, Cambridge, MA), monoclonal anti-human IL-1 recep-tor type II Ab (R & D Systems), and monoclonal anti-HAX Ab(BD Biosciences, San Jose, CA). Cells were incubated with theprimary Ab (5 �g�ml) or as controls with preimmune mouse IgG(5 �g�ml; Dako, Kyoto, Japan) for 1 h at 4°C. The primary Abwas detected by incubation with biotinylated anti-mouse IgG Abas the secondary Ab for 30 min at room temperature and thenincubated with Avidin�Biotin-HRP Complex (ABC; VectorLaboratories, Burlingame, CA). Cells were then stained byDAB-peroxidase substrate (Sigma). Hematoxylin was used fornuclear staining. The chamber slides were dried and examinedby light microscopy. Alternatively, after the treatment of the firstAb, cells were incubated with FITC- or Cy3-conjugated anti-mouse IgG Ab (Sigma). The chamber slides were washed threetimes and then mounted in 90% glycerol-PBS that contained0.1% paraphenylendiamine and 1% n-propylgalate. A fluores-cence image was obtained with fluorescence microscopy (Nikon,Tokyo, Japan).

Immunoprecipitation. SSc fibroblasts were cultured in DMEM(methionine�cystein-free) that contained 5% dialyzed FBS and100 �Ci�ml [35S]methionine�cystein (1 Ci � 37 GBq; Amersham

Bioscience, Buckinghamshire, U.K.) for 16 h. After a pulse, cellswere harvested and suspended in 3 ml of IP procedure (IPP)buffer (10 mM Tris, pH 8.0�0.5 M NaCl�0.1% Nonidet P-40�0.1mM PMSF�1 �g/ml leupeptin) and then sonicated on ice.Nuclear and cytosolic extracts were obtained together aftercentrifugation and were used for IP studies. A 40-�l volume ofprotein G-Sepharose was preincubated with rabbit anti-humanIL-1� Ab (100 ng; Genzyme, Cambridge, MA) or control rabbitIgG (100 ng; Dako) and was added to the extracts and rotatedfor 3 h at 4°C. Immunoprecipitates were washed three times withIPP buffer and then fractionated by 10% sodium dodecyl (lauryl)sulfate�polyacrylamide gel electrophoresis (SDS�PAGE) withmolecular weight markers-14C methylated protein (AmershamBioscience). Radiolabeled polypeptides were visualized byautoradiography.

Peptide Sequencing. Two specific bands (65 and 35 kDa) weresubjected to direct peptide sequencing. For sequencing, theproteins that were separated by SDS�PAGE were electro-phoretically transferred onto poly(vinylidene dif luoride)(PVDF) membrane (Bio-Rad, Richmond, CA). The PVDFmembrane was stained with Coomassie brilliant blue R-250, andeach band was excised and subjected to N-terminal amino acidsequence analysis (Procise 494 HT protein sequencing system;Applied Biosystems, Foster City, CA).

RT-PCR. Total RNA was extracted from cultured fibroblasts withTRIzol reagent (Invitrogen), and then 1 �g of total RNA wasreverse-transcribed into cDNA with SuperScript III (Invitrogen)according to the manufacturer’s instructions. Real-time RT-PCR was performed in triplicate with an ABI 7900HT system(Applied Biosystems) and a fluorescein-labeled (FAM-labeled)TaqMan gene expression assay kit (Applied Biosystems) forIL-1RII, HAX-1, and GAPDH as an endogenous control. Theresults were analyzed with SDS 2.1 software (Applied Biosys-tems). Those genes’ expressions were calculated from the accu-rate threshold cycle (Ct), which is the PCR cycle at which anincrease in fluorescein from TaqMan probes can first be de-tected above a baseline signal. The Ct values for GAPDH weresubstituted from the Ct values for IL-1RII and HAX-1 in eachwell to calculate �Ct. The triplicate �Ct values for each samplewere averaged.

Construction of Expression Plasmids and Transfection. The cDNAencoding human IL-1�, pre-IL-1�, and NTP-IL-1� were allisolated by PCR and subcloned into pcDNA4-V5 (Invitrogen).The cDNA encoding human IL-1RII was isolated by PCR andsubcloned into pcDNA3 (Invitrogen). For stable transfections,NIH 3T3 cells in 60-mm dishes (70% confluent) were incubatedwith 3 ml of Opti-MEM (Invitrogen) that contained 5 �g ofDNA and 18 �l of Lipofectamine 2000 (Invitrogen). After 5 h,3 ml of DMEM with 20% FBS was added. After 24 h, themedium was changed to DMEM with 10% FBS, followed by anadditional 24 h of culture. G418 (400 �g�ml) was added to theculture medium 48 h after transfection and kept for 15 days. TheG418-resistant colonies were harvested by gentle digestion withtrypsin, and cells were preserved in liquid N2 with Cellbanker(Mitsubishi Kagaku Iatron, Tokyo, Japan) until use.

Western Blotting. Confluent fibroblasts were maintained in aserum-free medium for 48 h. Cells were then trypsinized andwashed with PBS. Cell lysates were prepared from fibroblasts,including PBS that contained 0.1 mM PMSF and 1 �g�mlleupeptin by sonication on ice. The cell lysates were resolved in15% polyacrylamide gels under reducing conditions and trans-ferred to nitrocellulose membranes (Bio-Rad). The membraneswere incubated with the primary Abs for 1 h. Horseradishperoxidase-conjugated antimouse IgG Ab (Santa Cruz Biotech-

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nology, Santa Cruz, CA) was applied to the membrane andincubated for 1 h. The blot was developed by the enhancedchemiluminescence system (Amersham) and exposed on x-rayfilm. The primary Abs used in this experiment were monoclonalanti-human IL-1� Ab (R & D Systems), monoclonal anti-humanIL-1 receptor type II Ab (R & D Systems), monoclonalanti-V5 Ab (Invitrogen), and monoclonal anti-HAX Ab (BDBiosciences).

Bacterial Two-Hybrid System. Reagents and protocol were ob-tained from Stratagene (BacterioMatch two-hybrid system). Thepre-IL-1� protein was fused briefly to the full-length bacterio-phage � repressor protein (�cI) with pBT plasmid (Stratagene),which contained the N-terminal DNA-binding domain and theC-terminal dimerization domain. The target proteins (IL-1RIIand HAX-1) were fused to the N-terminal domain of the�-subunit of RNA polymerase with pTRG plasmid (Stratagene).The pre-IL-1� protein was tethered to the � operator sequenceupstream of the reporter promoter through the DNA-bindingdomain of �cI. When the pre-IL-1 and target proteins interact,they recruit and stabilize the binding of RNA polymerase at thepromoter and activate the transcription of a reporter gene, theAmpr gene. A second reporter gene, �-galactosidase, is ex-pressed from the same activatable promoter, which provides anadditional mechanism to validate the pre-IL-1� and targetproteins’ interaction. The suitable Escherichia coli host strain(XL1-Blue MRF� Kan) was transformed with the two plasmids.Blue colonies are positive in LB agar plates, including tetracy-cline, chloramphenicol, kanamycin, and X-gal.

Depletion of HAX-1 and IL-1RII by RNA Interference. The siRNAtarget-finder algorithm, which is available on the Ambion (Austin,TX) web site (www.ambion.com), was used to select 21 nucleotide

oligomers to be tested for RNA interference. We obtained a DNAfragment targeting the sequence in the ORF and a control with acorresponding random sequence. These two DNA fragments werecloned into pSilencer 3.1 H1-neo (Ambion), an siRNA-expressingvector, according to the manufacturer’s instructions. Target se-quences for siRNAs of HAX-1 and IL-1RII were selected to be5�-AACCCAAGGTTCCATAGTCCT-3� and 5�-AAGAA-GAGACACGGATGTGGG-3�, respectively. A random 21-nt se-quence as a control was generated that had the same numbersof nucleotides but did not display sequence identity with HAX-1and IL-1RII. Basic local alignment search tool analysis ensured thatsequence identity between a random nucleotide and homosapiencecDNA in the National Center for Biotechnology Informationdatabase was 15 nucleotides or fewer. The random sequences forHAX-1 and IL-1RII were 5�-AACCGCGAATCTCAT-AGTCCT-3� and 5�-AAGGAGAGCAGCGGATGTAAG-3�, re-spectively. The method for stable transfections was describedearlier.

Measurement of IL-6 and Procollagen Type I. Fibroblasts werecultured in 24-well culture plates with serum-free medium for48 h, and then the supernatants were collected and preserved at�30°C until use. IL-6 and procollagen type I were measured byusing commercial ELISA kits [R & D Systems and Takara Shuzo(Kyoto, Japan), respectively].

Statistical Analyses. The results of IL-6 and procollagen type Iconcentrations were shown as mean � SD, and comparisons ofdata were performed with Student’s t test. Differences wereconsidered to be significant at P � 0.05.

This work was supported by a research grant from the Ministry of Health,Labor, and Welfare (to Y. Kawaguchi) in Japan.

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