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Proc Natl Acad Sci U S A. Sep 26, 2006; 103(39): 14501–14506.
Published online Sep 13, 2006. doi:  10.1073/pnas.0603545103
PMCID: PMC1599989
Medical Sciences

Intracellular IL-1α-binding proteins contribute to biological functions of endogenous IL-1α in systemic sclerosis fibroblasts

Abstract

The aberrant production of precursor IL-1α (pre-IL-1α) in skin fibroblasts that are derived from systemic sclerosis (SSc) is associated with the induction of IL-6 and procollagen, which contributes to the fibrosis of SSc. However, little is understood about how intracellular pre-IL-1α regulates the expression of the other molecules in fibroblasts. We report here that pre-IL-1α can form a complex with IL-1α-binding proteins that is translocated into the nuclei of fibroblasts. Immunoprecipitation that used anti-human IL-1α Ab and 35S-labeled nuclear extracts of fibroblasts showed three specific bands (≈31, 35, and 65 kDa). The 31-kDa molecule was identified as pre-IL-1α, and the 35- and 65-kDa molecules might be pre-IL-1α-binding proteins. A partial sequencing for the 10 aa from the N-terminals of the molecules showed 100% homology for HAX-1 (HS1-associated protein X-1) and IL-1 receptor type II (IL-1RII). Suppression of the genes of HAX-1 or IL-1RII induced the inhibitory effects of IL-1 signal transduction, including production of IL-6 and procollagen, by fibroblasts. In particular, pre-IL-1α was not translocated into the nucleus by an inhibition of HAX-1. These findings reveal that nuclear localization of pre-IL-1α depends on the binding to HAX-1 and that biological activities might be elicited by the binding to both HAX-1 and IL-1RII in SSc fibroblasts.

Keywords: IL-1 receptor type II, HS1-associated protein X-1, fibrosis, collagen, IL-6

Systemic sclerosis (SSc) is a connective tissue disease of unknown etiology that is characterized by the fibrosis of systemic organs (1). Because skin thickening manifests in most patients, researchers have analyzed the molecular and biological functions of lesional skin fibroblasts that are derived from SSc patients (2). In previous reports we demonstrated that SSc fibroblasts expressed IL-1α mRNA constitutively and that aberrant production of precursor IL-1α (pre-IL-1α) contributed to skin fibrosis in SSc (35). IL-1α is a multifunctional molecule that is involved in a variety of inflammatory disorders, including sepsis, arthritis, myositis, psoriasis, periodontitis, and Alzheimer's disease (6). Pre-IL-1α is synthesized as a result of the transcription and translation of the IL1A gene. Under some circumstances, pre-IL-1α (31 kDa) is proteolytically cleaved to yield a mature form of IL-1α (17 kDa) (7). Because the N-terminal propiece of pre-IL-1α (NTP-IL-1α) contains a nuclear localization sequence (NLS), pre-IL-1α can be translocated into the nucleus, whereas mature IL-1α can be released from cells (8).

This pathway is complicated, however. The signal transduction of IL-1α is initiated by the binding of IL-1α (precursor or mature form) to cell-surface receptors on various cells (IL-1 receptor type I and IL-1 receptor accessory protein) (9, 10). The intracellular accumulation of pre-IL-1α in skin fibroblasts suggests an alternative pathway. Only a few studies, including ours, have reported the biological effects of intracellular IL-1α in fibroblasts and endothelial cells (1115). Although the precise pathway of signal transduction was not determined in those studies, the authors speculated that intracellular pre-IL-1α might exhibit a biological function directly and that the pathway of signal transduction might be distinct from the pathway that was mediated by binding the specific receptors. Our previous study (5) revealed that intracellular pre-IL-1α directly influenced the phenotype of SSc fibroblasts. These observations prompted us to explore the mechanism whereby intracellular pre-IL-1α exhibits its biological functions through the alternative pathway. In the present study we investigate the molecules that bind to pre-IL-1α in human fibroblasts and the effects of the IL-1α-binding proteins on nuclear localization and biological functions of IL-1α.

Results

Localization of Intracellular IL-1α in SSc Fibroblasts.

Although we previously demonstrated the nuclear localization of pre-IL-1α in SSc fibroblasts, we performed immunohistochemistry on five lines of SSc fibroblasts and three lines of normal fibroblasts. We visualized the signals of intracellular IL-1α in all five SSc fibroblast lines and did not detect them in the three normal fibroblast lines. A representative result of Cy3 staining is shown in Fig. 1. The specific signals were mostly distributed in the nucleus, consistent with our previous results (5).

Fig. 1.
Nuclear localization of IL-1α in SSc fibroblasts. SSc fibroblasts were cultured in plates on a four-chamber slide. Cells were fixed with 2% paraformaldehyde plus 0.1% Triton X-100. The primary Ab was monoclonal anti-human IL-1α Ab, which ...

Immunoprecipitation (IP).

To detect candidates of intracellular IL-1α-binding proteins, we used cell lysates of SSc fibroblasts and anti-IL-1α Ab to perform IP. As shown in Fig. 2, autoradiography indicated that the lengths of the specific bands were ≈31, 35, and 65 kDa. Columns 2 and 3 show representative data from IP that use cell lysates of SSc fibroblasts with anti-IL-1α Ab under a nonreducing and a reducing condition, respectively, and column 1 shows data that use cell lysates of SSc fibroblasts with rabbit IgG under a reducing condition. The 31-kDa band corresponded to the predicted pre-IL-1α in fibroblasts. Because the 35- and 65-kDa bands were also candidates of the intracellular IL-1α-binding proteins, we used a protein sequencer to partially analyze the N-terminals of these molecules. The 35-kDa molecule was homologous to HAX-1 (HS1-associated protein X-1; amino acid sequence MSLFDLFRGF), and the 65-kDa molecule was homologous to IL-1 receptor type II (IL-1RII; amino acid sequence FTLQPAAHTG). We observed no specific bands below the 30-kDa molecule (data not shown). Thus, we concluded that intracellular IL-1α was almost pre-IL-1α (31 kDa) in SSc fibroblasts, consistent with our previous studies (35).

Fig. 2.
Pre-IL-1α-binding proteins were detected by IP. Fibroblasts from SSc were cultured by using [35S]methionine/cystein for 16 h. After a pulse, cells were harvested and sonicated to extract nuclear and cytosolic proteins. IP was performed with cell ...

Expression of IL-1RII and HAX-1 in Fibroblasts.

To confirm the expression of IL-1RII and HAX-1 in SSc and normal fibroblasts, we used RT-PCR to analyze the expression of mRNA. The cDNA that was derived from five fibroblast lines of patients with SSc contained mRNA of both IL-1RII and HAX-1, but three normal fibroblast lines contained the HAX-1 mRNA alone (Table 1). Western blotting indicated that HAX-1 was expressed in SSc and normal fibroblasts, but we detected IL-1RII in SSc fibroblasts alone (Fig. 3). Immunocytochemical studies revealed different distributions of HAX-1 and IL-1RII between SSc and normal fibroblasts (Fig. 4). HAX-1 was localized in the nuclei and cytosol of SSc fibroblasts but in only the cytosol of normal fibroblasts. IL-1RII was localized in the nuclei and cytosol of SSc fibroblasts, and no fluorescent signal was detected in normal fibroblasts.

Fig. 3.
Western blot analysis of IL-1RII and HAX-1 in fibroblasts. Cell lysates were prepared from fibroblasts derived from systemic sclerosis (SSc) and a healthy donor (HC).
Fig. 4.
Cellular distribution of IL-1RII and HAX-1 by immunofluorescence staining. Fibroblasts were fixed by 2% paraformaldehyde plus 0.1% Triton X-100 and then reacted with anti-IL-1RII or anti-HAX-1 Ab. After they were treated with FITC-conjugated anti-mouse ...
Table 1.
Expression of IL-1RII and HAX-1 mRNA in cultured fibroblasts

Binding Capacities of IL-1RII to Pre-IL-1α in Fibroblasts.

To investigate the binding capacities of IL-1RII to pre-IL-1α in fibroblasts, we produced murine fibroblasts that were transfected with human pre-IL-1α, human IL-1α, or human NTP-IL-1α, which were cotransfected with human IL-1RII. We detected human IL-1RII, which was expressed by the pcDNA3 vector, in murine fibroblasts by anti-IL-1RII Ab (Fig. 5, second row). Because the three forms of human IL-1α were expressed as V5-tagged proteins in murine fibroblasts, these proteins were detected by anti-V5 Ab (Fig. 5, third row). Finally, after cell lysates from murine fibroblasts were immunoprecipitated with anti-IL-RII Ab, human IL-1α and human pre-IL-1α were detected by Western blotting with anti-V5 Ab (Fig. 5). These results indicate that intracellular IL-1RII binds to pre-IL-1α and mature IL-1α via the amino acid sequence from 113 to 271 aa.

Fig. 5.
A binding assay of pre-IL-1α and IL-1RII was performed with murine fibroblasts (NIH 3T3) transfected with human IL-1α and IL-1RII. The cDNA of human IL-1α (amino acids 113–271), pre-IL-1α (amino acids 1–271), ...

To confirm the interaction of IL-1α with IL-1RII and HAX-1, we fused cDNA that encodes three kinds of IL-1α to the λ repressor protein (λcI) of pBT plasmid and fused each target gene of IL-1RII and HAX-1 to the N-terminal domain of RNA polymerase of pTRG plasmid. We grew double transforming cells of pre-IL-1α with IL-1RII or HAX-1 in selection LB plates and had a β-galactosidase activity (Fig. 6). The cells transformed with NTP-IL-1α were positive only when cotransformed with HAX-1, and the cells transformed with IL-1α were positive only when cotransformed with IL-1RII. Previously, HAX-1 was reported to be associated with three sites of NTP-IL-1α (16). By considering all this evidence, we present a schematic of putative pre-IL-1α complex in Fig. 7.

Fig. 6.
A bacterial two-hybrid system was performed to confirm the interaction between pre-IL-1α and its binding proteins (IL-1RII and HAX-1). The IL-1α proteins were fused to the bacteriophage λ repressor protein by using pBT plasmid, ...
Fig. 7.
A putative structural component of the pre-IL-1α complex in fibroblasts. IL-1RII binds to the C-terminal domain of pre-IL-1α, and HAX-1 binds to the N-terminal domain in fibroblasts. However, unknown proteins, aside from these two, may ...

Functional Roles of IL-1RII and HAX-1 in the Signal Transduction of Pre-IL-1α.

To further investigate the roles of IL-1RII and HAX-1 as a pre-IL-1α-binding protein, we produced SSc fibroblasts that deplete IL-1RII or HAX-1 by means of RNA interference. IL-1RII and HAX-1 proteins were suppressed in all five lines of SSc fibroblasts transfected with a small siRNA-expressing vector. A representative result of Western blotting is shown in Fig. 8. We used five lines of SSc fibroblasts to conduct the experiments, and then we scanned each band on x-ray films on a scanning densitometer. We measured the intensity of each molecule by subtracting the intensity of background from that of the band. The mean ratio of specific/random siRNA was 0.06 in IL-1RII and 0.21 in HAX-1. An inhibition of IL-1RII did not affect the nuclear localization of pre-IL-1α, but inhibiting HAX-1 caused the nuclear staining of pre-IL-1α in SSc fibroblasts to disappear (Fig. 9). We used five different lines of SSc fibroblasts to confirm this result. We previously demonstrated that aberrant production of pre-IL-1α in the nucleus contributed to IL-6 and procollagen type I production in SSc fibroblasts. To explore the effects of IL-1RII and HAX-1 on IL-6 and procollagen type I production in SSc fibroblasts, we suppressed the production of both IL-6 and procollagen type I by the knockdown of IL-1RII and HAX-1 (Fig. 10A and B). The results indicate the mean of triplicate experiments that use five SSc fibroblasts and three normal fibroblasts.

Fig. 8.
Depletion of IL-1RII and HAX-1 by RNA interference. A DNA fragment that targeted the sequence in the ORFs of IL-1RII and HAX-1, and a control with a corresponding random sequence were obtained and were cloned into pSilencer 3.1H1-neo, an siRNA-expressing ...
Fig. 9.
Cell distribution of pre-IL-1α in SSc fibroblasts depleting IL-1RII or HAX-1. We obtained a DNA fragment targeting the sequence of IL-1RII or HAX-1, which was cloned into pSilencer 3.1 H1-neo, an siRNA-expressing vector. As a control, a scramble ...
Fig. 10.
IL-6 and procollagen type I C-peptide production decreases in SSc fibroblasts by the suppression of IL-1RII or HAX-1. Fibroblasts were cultured in serum-free media. After 48 h of culturing, commercial ELISA kits were used to measure IL-6 (A) and procollagen ...

Discussion

The results of the present study provide solid evidence that intracellular pre-IL-1α consists of a protein complex with IL-1RII and HAX-1 and that the formation of this complex is indispensable for pre-IL-1α-induced biological functions (IL-6 production and procollagen type I synthesis by fibroblasts). Our previous findings indicated that nuclear localization of pre-IL-1α plays a crucial role in the fibrogenic phenotype of skin fibroblasts derived from patients with SSc (35). The present study demonstrates the importance of the pre-IL-1α complex for the fibrogenic phenotype of SSc fibroblasts.

Early research indicated that IL-1α or pre-IL-1α is secreted from cells and exhibited an inflammatory response and immunity through the specific IL-1 receptors on the surface of targeted cells. However, intracellular pre-IL-1α has been shown to stimulate proliferation of renal fibroblasts (11) and to regulate the migration and the life span of endothelial cells (12), independent of secretion and cell-surface IL-1 receptors. Some researchers suggested that the nuclear localization sequence in the NTP-IL-1α molecule might be essential for the biological activity of intracellular IL-1α (11, 12). Recently, Buryskova et al. (13) observed that intracellular pre-IL-1α functionally activated transcription, interacting with histone acetyltransferase complexes. Werman et al. (14) reported that intracellular IL-1α is involved in the transcriptional activation of several proteins. Although IL-1α was traditionally understood to exhibit biological functions such as inflammation, autoimmunity, and fibrosis through IL-1 receptors on the cell surface, the abovementioned findings and this study strongly support the theory of a nuclear site of action for IL-1α.

Another important finding is a role of IL-1RII, which binds pre-IL-1α inside human fibroblasts that are derived from SSc. McMahon et al. (17) and Sims et al. (18) reported that IL-1RII is a cell-surface receptor on B lymphocytes and neutrophils with a binding affinity for IL-1α, pre-IL-1α, and IL-1β, but it is not capable of the signal transduction of IL-1 because of the lack of the endoplasmic domain. Our current results revealed that IL-1RII combined with pre-IL-1α plays a crucial role in the biological features of pre-IL-1α within SSc fibroblasts. We also found the differential expression of IL-1RII between SSc (n = 5) and normal fibroblasts (n = 3) at the cellular mRNA and protein levels. Constitutive expression of IL-1RII, as well as pre-IL-1α, may be an important phenotype of SSc fibroblasts, although the mechanisms whereby intracellular IL-1RII was highly expressed in SSc fibroblasts remain to be clarified.

Suzuki et al. (19) first identified the HAX-1 protein by screening the proteins that interact with HS1 (hematopoietic lineage cell-specific protein 1). HS1 is B cell-signaling protein and is one of the major substrates of the Src and Syk/Zap-70 kinases (20). The HS1 protein mainly exists in the cytoplasm and nucleus, and, when the molecule is associated with HAX-1, it moves to the mitochondrial membrane. HAX-1 also interacts with pre-IL-1α in human chondrocytes, although the biological properties for the complex of HAX-1 and pre-IL-1α have not been fully elucidated (16). The HAX-1 protein appears to be expressed ubiquitously in various normal tissues and to constitute the domain that is responsible for binding to the pre-IL-1α, HS1, cortactin, PKD2, EBNA-LP, Bcl-2, and HIV1 Vpr proteins (2123). The fact that HAX-1 interacts with a variety of structurally unrelated proteins suggests an essential function for HAX-1 that involves intracellular signaling and shuttling of various intracellular molecules. Our observations indicate the importance of HAX-1 for the nuclear localization of pre-IL-1α in fibroblasts. Posttranslational modifications such as phosphorylation and myristoylation of NTP-IL-1α are well recognized mechanisms that are involved in the transport of pre-IL-1α to the nucleus (24, 25). Notably, myristoylation occurs on lysine residues 82 and 83 of pre-IL-1α, located in the nuclear localization sequence (NLS). HAX-1 was associated with three segments of NTP-IL-1α, including the NLS segment (16), which suggests that the binding of HAX-1 with the NLS (KVLKKRR) of pre-IL-1α might facilitate the nuclear localization of the pre-IL-1α complex in fibroblasts. Taken together, the findings strengthen the conclusion that proteins associated with HAX-1 can shuttle between nuclear and cytoplasmic compartments.

A previous investigation looking for a nuclear target of pre-IL-1α revealed the interaction between pre-IL-1α and necdin by a yeast two-hybrid system (26). Necdin is a 47-kDa protein that functions as a cell-growth suppressor in a manner similar to that of the retinoblastoma tumor suppressor protein, Rb (27, 28). In our study, IP showed a faint band (≈47 kDa) that was subjected to N-terminal amino acid sequence analysis. However, we could not identify the molecule because of the small amount of peptide. Although we did not confirm that necdin was one of the intracellular pre-IL-1α-binding proteins, we did detect the expression of necdin in SSc and normal fibroblasts (data not shown). Moreover, the suppression of necdin with an RNAi method did not affect IL-6 and procollagen type I production in SSc fibroblasts (data not shown), which is inconsistent with the results 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).

Recent reports by Higgins et al. (29) and Kanangat et al. (30) demonstrated the biological functions of intracellular IL-1 receptor antagonist (icIL-1RA) in SSc fibroblasts. They indicated that icIL-1RA was overexpressed in SSc fibroblasts and that icIL-1RA was involved in the fibrogenic phenotype of SSc fibroblasts. Although we did not examine the expression of icIL-1RA in this study, icIL-1RA may have bound to intracellular IL-1RII that consisted of the pre-IL-1α complex. To determine whether icIL-1RA is the fourth component of the pre-IL-1α complex in SSc fibroblasts would have a potential role in delineating the molecular events of the fibrosis in SSc that are associated with the pre-IL-1α complex.

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

Materials and Methods

Cell Culture.

After providing informed consent, five female patients with SSc (median age 46) and three healthy female donors (median age 42) were enrolled in this study, which met the standards of our institutional review board. All patients were classified into diffuse cutaneous SSc according to the criteria of the American Rheumatism Association (31) and the classification of LeRoy et al. (32). Skin fibroblast lines were obtained from biopsied skin and explanted into tissue cultures. A murine fibroblast-like cell line, NIH 3T3, was also used in this study and was obtained from the American Tissue Culture Collection. The culture media consisted of DMEM (Sigma, St. Louis, MO) with 10% 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 third through 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 washed twice with cold PBS and fixed with 2% paraformaldehyde plus 0.1% Triton X-100 in PBS. The primary Abs used in this experiment were monoclonal anti-human IL-1α Ab (R & D Systems, Cambridge, MA), monoclonal anti-human IL-1 receptor type II Ab (R & D Systems), and monoclonal anti-HAX Ab (BD Biosciences, San Jose, CA). Cells were incubated with the primary Ab (5 μg/ml) or as controls with preimmune mouse IgG (5 μg/ml; Dako, Kyoto, Japan) for 1 h at 4°C. The primary Ab was detected by incubation with biotinylated anti-mouse IgG Ab as the secondary Ab for 30 min at room temperature and then incubated with Avidin/Biotin-HRP Complex (ABC; Vector Laboratories, Burlingame, CA). Cells were then stained by DAB-peroxidase substrate (Sigma). Hematoxylin was used for nuclear staining. The chamber slides were dried and examined by light microscopy. Alternatively, after the treatment of the first Ab, cells were incubated with FITC- or Cy3-conjugated anti-mouse IgG Ab (Sigma). The chamber slides were washed three times and then mounted in 90% glycerol-PBS that contained 0.1% paraphenylendiamine and 1% n-propylgalate. A fluorescence image was obtained with fluorescence microscopy (Nikon, Tokyo, Japan).

Immunoprecipitation.

SSc fibroblasts were cultured in DMEM (methionine/cystein-free) that contained 5% dialyzed FBS and 100 μCi/ml [35S]methionine/cystein (1 Ci = 37 GBq; Amersham Bioscience, Buckinghamshire, U.K.) for 16 h. After a pulse, cells were 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-40/0.1 mM PMSF/1 μg/ml leupeptin) and then sonicated on ice. Nuclear and cytosolic extracts were obtained together after centrifugation and were used for IP studies. A 40-μl volume of protein G-Sepharose was preincubated with rabbit anti-human IL-1α Ab (100 ng; Genzyme, Cambridge, MA) or control rabbit IgG (100 ng; Dako) and was added to the extracts and rotated for 3 h at 4°C. Immunoprecipitates were washed three times with IPP buffer and then fractionated by 10% sodium dodecyl (lauryl) sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) with molecular weight markers-14C methylated protein (Amersham Bioscience). Radiolabeled polypeptides were visualized by autoradiography.

Peptide Sequencing.

Two specific bands (65 and 35 kDa) were subjected to direct peptide sequencing. For sequencing, the proteins that were separated by SDS/PAGE were electrophoretically transferred onto poly(vinylidene difluoride) (PVDF) membrane (Bio-Rad, Richmond, CA). The PVDF membrane was stained with Coomassie brilliant blue R-250, and each band was excised and subjected to N-terminal amino acid sequence analysis (Procise 494 HT protein sequencing system; Applied Biosystems, Foster City, CA).

RT-PCR.

Total RNA was extracted from cultured fibroblasts with TRIzol reagent (Invitrogen), and then 1 μg of total RNA was reverse-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) for IL-1RII, HAX-1, and GAPDH as an endogenous control. The results were analyzed with SDS 2.1 software (Applied Biosystems). Those genes' expressions were calculated from the accurate threshold cycle (Ct), which is the PCR cycle at which an increase in fluorescein from TaqMan probes can first be detected above a baseline signal. The Ct values for GAPDH were substituted from the Ct values for IL-1RII and HAX-1 in each well to calculate ΔCt. The triplicate ΔCt values for each sample were averaged.

Construction of Expression Plasmids and Transfection.

The cDNA encoding human IL-1α, pre-IL-1α, and NTP-IL-1α were all isolated by PCR and subcloned into pcDNA4-V5 (Invitrogen). The cDNA encoding human IL-1RII was isolated by PCR and subcloned into pcDNA3 (Invitrogen). For stable transfections, NIH 3T3 cells in 60-mm dishes (70% confluent) were incubated with 3 ml of Opti-MEM (Invitrogen) that contained 5 μg of DNA and 18 μl of Lipofectamine 2000 (Invitrogen). After 5 h, 3 ml of DMEM with 20% FBS was added. After 24 h, the medium was changed to DMEM with 10% FBS, followed by an additional 24 h of culture. G418 (400 μg/ml) was added to the culture medium 48 h after transfection and kept for 15 days. The G418-resistant colonies were harvested by gentle digestion with trypsin, and cells were preserved in liquid N2 with Cellbanker (Mitsubishi Kagaku Iatron, Tokyo, Japan) until use.

Western Blotting.

Confluent fibroblasts were maintained in a serum-free medium for 48 h. Cells were then trypsinized and washed with PBS. Cell lysates were prepared from fibroblasts, including PBS that contained 0.1 mM PMSF and 1 μg/ml leupeptin by sonication on ice. The cell lysates were resolved in 15% polyacrylamide gels under reducing conditions and transferred to nitrocellulose membranes (Bio-Rad). The membranes were incubated with the primary Abs for 1 h. Horseradish peroxidase-conjugated antimouse IgG Ab (Santa Cruz Biotechnology, Santa Cruz, CA) was applied to the membrane and incubated for 1 h. The blot was developed by the enhanced chemiluminescence system (Amersham) and exposed on x-ray film. The primary Abs used in this experiment were monoclonal anti-human IL-1α Ab (R & D Systems), monoclonal anti-human IL-1 receptor type II Ab (R & D Systems), monoclonal anti-V5 Ab (Invitrogen), and monoclonal anti-HAX Ab (BD Biosciences).

Bacterial Two-Hybrid System.

Reagents and protocol were obtained from Stratagene (BacterioMatch two-hybrid system). The pre-IL-1α protein was fused briefly to the full-length bacteriophage λ repressor protein (λcI) with pBT plasmid (Stratagene), which contained the N-terminal DNA-binding domain and the C-terminal dimerization domain. The target proteins (IL-1RII and HAX-1) were fused to the N-terminal domain of the α-subunit of RNA polymerase with pTRG plasmid (Stratagene). The pre-IL-1α protein was tethered to the λ operator sequence upstream of the reporter promoter through the DNA-binding domain of λcI. When the pre-IL-1 and target proteins interact, they recruit and stabilize the binding of RNA polymerase at the promoter and activate the transcription of a reporter gene, the Ampr gene. A second reporter gene, β-galactosidase, is expressed from the same activatable promoter, which provides an additional mechanism to validate the pre-IL-1α and target proteins' 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 tetracycline, chloramphenicol, kanamycin, and X-gal.

Depletion of HAX-1 and IL-1RII by RNA Interference.

The siRNA target-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 DNA fragment targeting the sequence in the ORF and a control with a corresponding random sequence. These two DNA fragments were cloned into pSilencer 3.1 H1-neo (Ambion), an siRNA-expressing vector, according to the manufacturer's instructions. Target sequences for siRNAs of HAX-1 and IL-1RII were selected to be 5′-AACCCAAGGTTCCATAGTCCT-3′ and 5′-AAGAAGAGACACGGATGTGGG-3′, respectively. A random 21-nt sequence as a control was generated that had the same numbers of nucleotides but did not display sequence identity with HAX-1 and IL-1RII. Basic local alignment search tool analysis ensured that sequence identity between a random nucleotide and homosapience cDNA in the National Center for Biotechnology Information database was 15 nucleotides or fewer. The random sequences for HAX-1 and IL-1RII were 5′-AACCGCGAATCTCATAGTCCT-3′ and 5′-AAGGAGAGCAGCGGATGTAAG-3′, respectively. The method for stable transfections was described earlier.

Measurement of IL-6 and Procollagen Type I.

Fibroblasts were cultured in 24-well culture plates with serum-free medium for 48 h, and then the supernatants were collected and preserved at −30°C until use. IL-6 and procollagen type I were measured by using commercial ELISA kits [R & D Systems and Takara Shuzo (Kyoto, Japan), respectively].

Statistical Analyses.

The results of IL-6 and procollagen type I concentrations were shown as mean ± SD, and comparisons of data were performed with Student's t test. Differences were considered to be significant at P < 0.05.

Acknowledgments

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

Abbreviations

Ct
threshold cycle
HAX-1
HS1-associated protein X-1
icIL-1RA
intracellular IL-1 receptor antagonist
IL-1RII
IL-1 receptor type II
IP
immunoprecipitation
NTP-IL-1α
N-terminal propiece of pre-IL-1α
pre-IL-1α
precursor IL-1α
SSc
systemic sclerosis.

Footnotes

The authors declare no conflict of interest.

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

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