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Autocrine Induction of the Human Pro-IL-1 Gene Promoter by IL-1 in Monocytes

Yoko Toda^*, Junichi Tsukada^1,*, Masahiro Misago^*,, Yoshihiko Kominato, Philip E. Auron and Yoshiya Tanaka^*

^* First Department of Internal Medicine, School of Medicine, and School of Health Sciences, University of Occupational and Environmental Health, Kitakyushu, Japan; Department of Legal Medicine, Toyama Medical and Pharmaceutical University, Toyama, Japan; and Department of Medicine, New England Baptist Bone and Joint Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02181

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
IL-1 is produced primarily by activated monocytes/macrophages. We report in this study that IL-1 induces the human pro-IL-1 (IL1B) gene promoter in human THP-1 monocytic cells. The -131 to +12 minimal IL1B promoter was induced by IL-1 in a dose-dependent manner. The promoter possesses two important transcription factor binding motifs, one for an ETS family transcription factor Spi-1 (PU.1), and the other a binding site for NF-IL6 (CCAAT/enhancer binding protein ). Autocrine promoter activity was completely inhibited by mutation of the Spi-1 site. Mutation of the NF-IL6 binding motif caused partial loss of activity. EMSAs using THP-1 cell nuclear extracts indicated that IL-1 significantly induced Spi-1 binding to its target site within the IL1B promoter that was maximal at 1 h after stimulation, correlating with the kinetics of IL-1 induction. The importance of Spi-1 was supported by our observation that Spi-1-deficient EL4 thymocytes exhibited IL-1-induced activity only after transfection with a Spi-1 expression vector. Moreover, TNFR-associated factor 6 also required Spi-1 to activate the promoter. Transfection studies using Spi-1 mutant constructs showed that the TATA-binding protein binding and glutamine-rich domains of Spi-1 were important for IL-1 induction, whereas LPS induction required the proline, glutamic acid, serine, and threonine-rich domain containing serine 148 as well as the TATA-binding protein and glutamine-rich domains. We conclude that the IL1B promoter is an IL-1-responsive sequence as a result of its ability to bind Spi-1 in response to IL-1.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Interleukin-1 is a multifunctional cytokine that mediates a wide spectrum of inflammatory, metabolic, physiologic, hematopoietic, and immunological processes (1, 2, 3). A dramatic increase in IL-1 production occurs in response to various stimuli, including LPS, PMA, and cytokines. Some cells, but not exclusively tumor cells, produce cytokines that activate receptors on their own cell surface. It is well known that IL-1 stimulates its own gene expression and synthesis in vascular smooth muscle cells (4), endothelial cells (5), and monocytes (6, 7). Increased IL-1 production has been shown in various human diseases (1, 2, 3). These findings suggest that autoinduction of IL-1 may contribute to some pathologic processes via self-amplification of gene expression. IL-1 is a principal mediator in the pathogenesis of rheumatoid arthritis. It is involved in the mechanisms that result in progressive joint destruction in rheumatoid arthritis. Ghivizzani et al. (8) have reported that expression of human IL-1 following gene transfer to rabbit knee joints results in a severe aggressive form of arthritis with elevated levels of rabbit IL-1 and TNF- in rabbit synovial fluid. Moreover, IL-1 is an autocrine growth factor for human acute myeloid leukemia cells (9, 10). It is noteworthy that in some cases IL-1 production in acute myeloid leukemia cells was partially inhibited by IL-1R antagonist (11).

IL-1 is most abundantly expressed in activated monocytes/macrophages. Production of IL-1 is tightly regulated in monocytes/macrophages. The human pro-IL-1 gene (referred to here by its genomic locus name, IL1B) encoding pro-IL-1, a 31-kDa IL-1 precursor protein, is normally silent but is rapidly transcribed in competent cells upon stimulation (12, 13). The best-characterized stimulus that triggers IL-1 production is LPS. It was previously reported that human blood monocytes possess receptors for IL-1 (14). IL-1 production in monocytes has been demonstrated to be induced in response to IL-1 treatment (6, 7). However, the mechanism by which IL-1 itself activates the IL1B gene remains unclear.

Monocyte/macrophage-specific expression of the IL1B gene depends upon its promoter located between positions -131 and +12 (15, 16). The IL1B promoter contains two important transcription factor binding motifs: one is a binding site for NF-IL6, which is the form of the CCAAT/enhancer binding protein (C/EBP)² of the basic leucine zipper family; and the other for Spi-1 (PU.1), a myeloid and B cell-specific winged helix-turn-helix transcription factor that belongs to the ETS family of proteins.

In the present study, we examined the promoter activity of the IL1B gene in transiently transfected human THP-1 monocytic cells and demonstrated that IL-1 induced the IL1B promoter in a dose-dependent manner. Mutation of the Spi-1 binding site within the IL1B gene promoter completely inhibited IL-1-induced promoter activity, whereas mutation of the NF-IL6 site resulted in a partial loss of promoter activity. The results of EMSA using nuclear extracts prepared from IL-1-treated THP-1 monocytes showed that IL-1 induced binding of Spi-1 to the IL1B target site. The importance of Spi-1 was further supported by our finding that IL-1 induction of the IL1B promoter was not observed in Spi-1-deficient EL4 cells, but was detected in EL4 cells carrying a Spi-1 expression vector. TNFR-associated factor 6 (TRAF6) also required Spi-1 expression in EL4 cells to activate the promoter. Transfection studies using various activation domain deletions of the Spi-1 cDNA revealed that IL-1 induction of the IL1B promoter depends upon the TATA-binding protein (TBP) binding and glutamine-rich (Q) domains of the Spi-1 protein, whereas activation by LPS requires the proline, glutamic acid, serine, and threonine-rich (PEST) domain containing serine 148 as well as the TBP and Q domains. Based on these results, we propose that the IL1B promoter is a IL-1-responsive sequence as a result of its ability to bind Spi-1 in response to IL-1.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Endotoxin tests

Very low concentrations of endotoxin induce IL1B gene transcription in THP-1 monocytes, as described previously (17). Therefore, all materials and solutions including RPMI 1640 medium, FBS (Equitech-bio, Ingram, TX), and human rIL-1 were tested for endotoxin by the Limulus amebocyte lysate test, as described previously (15). In particular, IL-1 used in the present study contained <0.003 ng/mg endotoxin. Moreover, sterile irrigation water and disposable sterile pipettes and tubes were used to reduce endotoxin levels. Basal IL1B gene activation was avoided by culturing cells in endotoxin-free medium.

rIL-1 protein and other reagents

Human rIL-1 protein (1 x 10⁸ U/mg as assayed by a thymocyte proliferation assay) was provided by Otsuka Pharmacia (Fukuoka, Japan). Human rIL-1 protein had a specific activity of 1 x 10⁸ U/mg (Dainippon Pharmacia, Osaka, Japan). Anti-Spi-1 Ab and anti-Oct-1 Ab were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Spi-1 Ab was raised against a synthetic peptide corresponding to amino acids 251–271 mapping at the carboxyl terminus of Spi-1 of mouse origin. Anti-Oct-1 Ab was raised against a synthetic peptide corresponding to amino acids 723–743 mapping at the carboxyl terminus of Oct-1 of human origin. The two Abs did not cross-react with other transcription factors. LPS (Escherichia coli O55:B5), PMA, rabbit anti-human IL-1 Ab, and rabbit anti-human IL-1 Ab were purchased from Sigma-Aldrich (St. Louis, MO). One milligram of rabbit anti-human IL-1 Ab neutralizes a minimum of 7,000 U of human IL-1. One milligram of rabbit anti-human IL-1 Ab neutralizes a minimum of 35,000 U of human IL-1.

Cells

The human THP-1 monocytic cell line (JCRB0112) was purchased from Health Science Research Resources Bank (Osaka, Japan). The mouse EL4 thymoma cell line was kindly provided by the Chemo-Sero-Therapeutic Research Institute (Kumamoto, Japan). Cells were carefully maintained in endotoxin-free complete RPMI 1640 medium supplemented with 10% FBS. Cells were split at a 1/3 dilution every 3 or 4 days to avoid overcrowding and were further split at 1/2 on the day before transfection or preparation for nuclear extracts.

Plasmids

Human IL1B genomic DNA fragments were derived from clone BDC454 (18). We used identical sequence numbering to that described previously (15). The construct, 3MEHT, contained the IL1B promoter HT sequence (16). The HT sequence (construct 3MEHT) located between positions -131 and +12 was cloned into the chloramphenicol acetyltransferase (CAT) gene plasmid vector pA10CAT3ME (3ME). Mutations of the Spi-1 and NF-IL6 sites were the same as those reported previously (16). These mutations were verified by sequencing. MHC/fosCAT contains three tandem repeats of the NF-B binding site of the MHC class I gene enhancer as described previously (19, 20). The series of Spi-1 pECE expression vectors consisting of a wild-type Spi-1 cDNA and deletion constructs were gifts from Dr. R. A. Maki (Burnham Institute, La Jolla, CA) (21). Expression vectors for the full-length NF-IL6 (pcNF-IL6) and a truncated NF-IL6 with a deletion of the internal SplI/SplI fragment (amino acid sequence between residues 41 and 205) (pcmNF-IL6(spl)) were generated by inserting the respective coding regions into pcDNA1 (Invitrogen, Carlsbad, CA) (19). A TRAF6 expression vector, pcTRAF6, was constructed by inserting the TRAF6 cDNA into the pcDNA3.1 expression vector (Invitrogen).

Transfection and CAT assay

THP-1 cells and EL4 cells were transfected by the DEAE-dextran method as described previously (15, 22). This technique was used because, unlike electroporation, it did not generate induction of the endogenous IL1B gene. Cells (1 x 10⁷ cells per plate) were transfected with 1018 µg of plasmids. After transfection, the cells were left untreated or were treated with IL-1 (110 ng/ml for THP-1 cells and 2 ng/ml for EL4 cells) for 24 h. CAT assays were performed using the liquid scintillation method as described previously (15), except using Pica Gene cell lysis buffer (Toyo Ink, Tokyo, Japan). CAT activities were determined by calculating slopes from plots of time vs cpm within a linear range of the response.

Nuclear extracts and EMSA

Nuclear extracts were prepared from THP-1 monocytes and EL4 thymocytes as reported previously (15). Protein concentrations of extracts were determined using the Bio-Rad protein assay kit (Melville, NY). Oligonucleotides were labeled by filling in 3' recessed ends with the DNA polymerase Klenow fragment and [-³²P]dNTP (Amersham Life Science, Little Chalfont, Buckinghamshire, U.K.). Binding reactions were conducted as described previously, followed by analysis on a 4% polyacrylamide gel using 0.5x TBE buffer (89 mM Tris-borate and 2.5 mM EDTA) as the running buffer (16).

RT-PCR

THP-1 cells were harvested after treatment with 10 ng/ml of IL-1, and total RNA was extracted by ISOGEN RNA extraction kit (Nippon Gene, Tokyo, Japan). After spectrophotometric quantification, 200 ng of total RNA was used along with a reverse transcriptase RNA PCR kit, Access RT-PCR System (Promega, Madison, WI) according to the manufacturer’s instructions. An aliquot of the PCR mixture was subjected to electrophoresis in 2% agarose gel. PCR primers were synthesized as follows: human IL-1 sense, 5'-CAGAGAGTCCTGTGCTGAAT-3'; human IL-1 antisense, 5'-GTAGGAGAGGTCAGAGAGGC-3' (23); -actin sense, 5'-TCATGAAGTGTGACGTTGACATCCGT-3'; and -actin antisense, 5'-CCTAGAAGCATTTGCGGTGCACGATG-3'.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Autocrine induction of the IL1B promoter by IL-1

We previously demonstrated that monocyte-specific expression of the human IL1B gene depends upon the IL1B promoter, located between positions -131 and +12 of the gene (15, 16). IL-1 has been reported to induce production of G-CSF in human THP-1 monocytic cells (24). In the present study, we examined the effect of IL-1 on IL1B promoter activity. The -131 to +12 IL1B promoter element (HT fragment) was introduced into the 3ME CAT vector and assayed for IL-1-induced CAT activity following transfection into THP-1 cells. As shown in Fig. 1, IL-1 induced IL1B promoter activity in a dose-dependent manner. When 10 ng/ml of IL-1 was used, an 5-fold increase in activity over control was observed. 3ME vector, in contrast to 3MEHT, did not show any increased CAT activity following IL-1 treatment. These results indicate that IL-1 induces the IL1B promoter in THP-1 monocytic cells.

View larger version (19K): [in this window] [in a new window]   FIGURE 1. IL-1 dose-dependently induces the IL1B promoter activity in THP-1 monocytes. Ten micrograms of 3MEHT (•) or 3ME () was transfected into THP-1 cells. After transfection, cells were treated with various concentrations of IL-1 or left untreated. The CAT activity was calculated as described in Materials and Methods. Data represent the mean ± SD of three experiments. The CAT data were normalized to the average activity elicited by the 3MEHT construct in the absence of IL-1.

The -50 to -39 Spi-1 binding site is essential for IL-1 induction of the IL1B promoter

The IL1B promoter HT fragment contains two important transcription factor binding motifs: one is a binding site for NF-IL6 and the other is a binding site for Spi-1 (Fig. 2A). In the present study, two distinct mutated CAT constructs, 3MEHTmSpi-1 and 3MEHTmNF-IL6, were used to examine specific sequence requirements for IL-1 induction in THP-1 monocytic cells. Specific nucleotide substitutions (Fig. 2A) were introduced into either the -50 to -39 Spi-1 binding site (HTmSpi-1) or the -91 to -83 NF-IL6 site (HTmNF-IL6). As shown in Fig. 2B, mutation of the Spi-1 site almost completely abolished IL-1-induced promoter activity. These results reveal that the Spi-1 site is essential for IL-1 induction of the IL1B promoter. In contrast, the -91 to -83 NF-IL6 binding motif mutation (3MEHTmNF-IL6) caused only a partial loss of activity (60% of the wild-type 3MEHT), suggesting that the NF-IL6 site is not essential for IL-1 induction but is important for maximal transcriptional activity. This argument is supported by our finding in EMSA experiments that the mutation of the -91 to -83 NF-IL6 binding motif completely abolished binding of rNF-IL6 to the IL1B promoter HD fragment (Fig. 2C, lanes 1–3). HDmNF-IL6, which was generated by restriction endonuclease digestion, was identical to the wild-type HD but contained nucleotide substitutions, mNF-IL6, within the -91 to -83 region. Moreover, when the binding affinity of rSpi-1 for the wild-type DT was compared with that for DTmSpi-1, the mutation significantly inhibited binding of rSpi-1 (Fig. 2C, lanes 4–6).

View larger version (23K): [in this window] [in a new window]   FIGURE 2. Mutation of the -50 to -39 Spi-1-binding site abrogates IL-1-induced promoter activity. A, The schema shows the wild-type IL1B promoter HT fragment and two distinct mutated HT (HTmNF-IL6 and HTmSpi-1). As indicated by arrows, these mutations are located at specific sites known to be critical for NF-IL6 and Spi-1 binding, respectively. B, CAT reporters were transfected into THP-1 cells and treated with 2 ng/ml IL-1. The CAT data were normalized to the average activity elicited by IL-1-induced wild-type 3MEHT. Data represent the mean ± SD of three experiments. C, EMSAs were performed with rNF-IL6 and Spi-1 proteins. HD and DT fragments (A) were used as radiolabeled probes. Unlabeled competitor DNAs were used at a 30-fold molar excess over the radiolabeled probes.

IL-1 induces binding of Spi-1 to the IL1B promoter

Our mutation studies showed that IL-1-induced transcriptional activation of the IL1B promoter requires the -50 to -39 Spi-1 binding motif. To examine binding of specific protein to the Spi-1 site, EMSA studies were performed using a radiolabeled DT probe containing the -50 to -39 Spi-1 binding site (Fig. 2A). Nuclear extracts were prepared from THP-1 cells treated with IL-1. As shown in Fig. 3, A and B, THP-1 nuclear extracts generated a DNA-protein complex (Fig. 3, arrow), which comigrated with in vitro expressed Spi-1 protein (Fig. 3, lanes 1). Although untreated THP-1 nuclear extract showed a constitutive binding activity (Fig. 3A, lane 2), the complex formation was markedly enhanced following treatment with IL-1. The intensities of the complex reached a maximum at 1 h after stimulation (Fig. 3A, lane 3) and returned to a basal level 3 h after treatment (Fig. 3A, lane 5). The complex was competed for by a 30-fold molar excess of unlabeled DT fragment itself (Fig. 3B, lane 3). In contrast, DT containing site-specific mutation of the Spi-1 binding site (DTmSpi-1) did not compete for the protein-DT complex (Fig. 3B, lane 4). Furthermore, the complex was abrogated and supershifted by the addition of anti-Spi-1 Ab (Fig. 3B, lane 5), whereas it was not recognized by anti-Oct-1 Ab (Fig. 3B, lane 6). These results demonstrate that the IL-1-induced DT-protein complex contains Spi-1 protein.

View larger version (31K): [in this window] [in a new window]   FIGURE 3. Spi-1 binds to the IL1B promoter through IL-1 stimulation. DT (Fig. 2A) oligonucleotide was used as a radiolabeled probe. Lanes 1, rSpi-1 protein obtained by in vitro translation (rSpi-1) was used as a control. The arrows locate the mobility of Spi-1. A, Nuclear extracts were derived from THP-1 cells untreated (lane 2) or treated with IL-1 for the time indicated over lanes (lanes 3-5). B, Nuclear extract was prepared from THP-1 cells treated with IL-1 for 1 h (lanes 2–6). Unlabeled competitor DNAs were used at a 30-fold molar excess over the radiolabeled DT probe (lanes 3 and 4). In supershift experiments, nuclear extracts were preincubated with either anti-Spi-1 Ab or anti-Oct-1 Ab at room temperature for 15 min (lanes 5 and 6).

Expression of Spi-1 in EL4 cells mediates IL-1-induced IL1B promoter activation

To clarify the functional involvement of Spi-1 protein in IL-1 induction of the IL1B promoter, various amounts of a Spi-1 expression vector, pECE Spi-1, were cotransfected into EL4 thymoma cells along with the IL1B promoter CAT reporter, 3MEHT. Following cotransfection, cells were treated with 2 ng/ml IL-1 or left untreated, and assayed for CAT activity. Untransfected EL4 cells do not express Spi-1 protein (Fig. 5C, lane 2). As shown in Fig. 4, IL-1-induced activity in EL4 cells was increased in a Spi-1 dose-dependent manner. In the presence of 4 µg of pECE Spi-1, IL-1 treatment resulted in an 3-fold increase in activity over control without IL-1 treatment. In contrast, IL-1 failed to augment the IL1B promoter activity in Spi-1-deficient EL4 cells. These results demonstrate that Spi-1 functions as a transcriptional activator that is essential for IL-1 induction.

View larger version (28K): [in this window] [in a new window]   FIGURE 5. TBP binding and Q domains of Spi-1 are required for IL-1 to induce the IL1B promoter. A, Schematic diagram of Spi-1 functional domains previously identified and the regions contained within various Spi-1 expression vectors. B, Various Spi-1 expression vectors (4 µg) as depicted in A were cotransfected into either EL4 thymoma or THP-1 monocyte cells along with 3MEHT CAT reporter (10 µg). Following transfection, EL4 cells were treated with 2 ng/ml IL-1 or left untreated. THP-1 cells were treated with either 100 ng/ml of LPS or 50 ng/ml of PMA. The CAT data were normalized to the average activity elicited by the IL-1-induced 3MEHT construct in wild-type (WT) Spi-1-expressing EL4 cells. Data represent the mean ± SD of three experiments. C, DT was used as a radiolabeled probe. Nuclear extracts were prepared from EL4 thymoma cells carrying various Spi-1 expression vectors as indicated over lanes. At 24 h after transfection, the cells were stimulated by 2 ng/ml IL-1 for 1 h. D, The effects of endogenous IL-1 expression on LPS- or PMA-induced IL1B promoter activity were investigated. Spi-1 expression vectors (4 µg) were cotransfected into THP-1 cells along with 3MEHT CAT reporter (10 µg). As a control study, MHC/fosCAT (10 µg) was transfected into EL4 cells. Following transfection, the cells were treated as indicated. LPS, PMA, IL-1, and Abs were used at the following concentrations: LPS, 100 ng/ml; PMA, 50 ng/ml; IL-1, 1 ng/ml; IL-1, 1 ng/ml; anti-IL-1 Ab, 10 µg/ml; and anti-IL-1 Ab, 2 µg/ml. The total amount of added IgG was kept constant by addition of control IgG. The CAT data were normalized to the average activity elicited by the MHC/fosCAT in unstimulated EL4 cells or PMA-induced 3MEHT in THP-1 cells. Data represent the mean ± SD of three experiments.

View larger version (16K): [in this window] [in a new window]   FIGURE 4. IL-1 induces the IL1B promoter in Spi-1-expressing EL4 cells. Increasing amounts of a Spi-1 expression vector, pECE Spi-1 were cotransfected into EL4 thymoma cells along with 3MEHT reporter (10 µg). The total amount of transfected DNA was kept constant (14 µg) by addition of control vector. At 24 h after transfection, the cells were treated with 2 ng/ml IL-1 (•) or left untreated (). The CAT data were normalized to the average activity elicited by the IL-1-induced 3MEHT construct in the absence of expression vector cotransfection. Data represent the mean ± SD of three experiments.

IL-1 induction depends on a Q domain and a TBP binding domain of Spi-1 protein

Spi-1 protein contains at least three independent transcriptional activation domains: a TBP binding region, a Q domain, and a PEST region (Fig. 5A) (21). In the present study, to determine the domain of Spi-1 protein necessary for IL-1 induction of the IL1B promoter, various deletion mutant Spi-1 proteins were assayed in transient transfection studies using EL4 cells. As shown in Fig. 5B, removal of the PEST sequence did not reduce IL-1-dependent activity, indicating that the PEST domain was dispensable for IL-1 induction of the IL1B promoter. This is consistent with our observation that mutation of a serine to alanine at codon 148 (S148A) also failed to inhibit the IL-1-induced activity. However, deletion of either the Q domain (75/100) or the TBP binding region (8/32) markedly inhibited the IL-1-inducible activity. This argument is further supported by our EMSA data using nuclear extracts prepared from EL4 cells transiently transfected with expression vectors for the mutant Spi-1 proteins. As shown in Fig. 5C, the relative expression levels of the mutant Spi-1 proteins in EL4 cells were almost the same. Moreover, when the various deletion mutant Spi-1 proteins were expressed in THP-1 monocytes, PMA induction also did not require serine 148. In contrast, S148A markedly inhibited the ability of Spi-1 to transactivate the IL1B promoter in the presence of LPS.

We further performed transient transfection studies using neutralizing Abs against IL-1 and IL-1 to elucidate the effect of endogenous IL-1 expression on activation of the IL1B promoter by LPS or PMA. Neutralizing activities of the Abs were assessed in transfection studies using EL4 cells and MHC/fosCAT vector containing tandem repeats of NF-B binding site. As shown in Fig. 5D, anti-IL-1 Ab almost completely inhibited IL-1-induced activity for MHC/fosCAT. IL-1-induced CAT activity for MHC/fosCAT was also neutralized by the addition of anti-IL-1 Ab. However, neither LPS induction nor PMA induction of the IL1B promoter in THP-1 cells was inhibited by the addition of a mixture of anti-IL-1 Ab and anti-IL-1 Ab, showing that endogenous IL-1 expression has no effect on IL1B promoter activity induced by either LPS or PMA. Spi-1 protein activation by either LPS or PMA was not also inhibited by the addition of the Abs. In particular, the addition of the Abs failed to affect activation of Spi-1 S148 mutant by PMA.

TRAF6-mediated activation of the IL1B promoter requires Spi-1

The IL-1 signal transduction is initiated by the association of IL-1 with the three extracellular Ig domains of IL-1 type I receptor, which, in turn, results in the association of IL-1R accessory protein (2, 25, 26, 27). This signaling complex recruits the adapter protein MyD88, which mediates the interaction of the IL-1R-associated kinases (IRAK). IRAK recruits TRAF6 to the activated IL-1 type I receptor. TRAF6 as well as MyD88 and IRAK are critical signal transducers for IL-1 (28, 29). In the present study, a TRAF6 expression vector, pcTRAF6, and a Spi-1 expression vector, pECE Spi-1, alone or in combination was cotransfected into EL4 cells along with the IL1B promoter CAT reporter 3MEHT and assayed for TRAF6-induced promoter activity in the presence and absence of Spi-1. As shown in Fig. 6, transfection of pcTRAF6 did not affect IL1B promoter activity in the absence of Spi-1. Similar results were observed when the cells were treated with IL-1. However, when pECE Spi-1 was cotransfected into EL4 cells with pcTRAF6, transfection of pcTRAF6 significantly induced the IL1B promoter. Moreover, treatment of Spi-1-expressing EL4 with IL-1 enhanced the TRAF6-induced promoter activity. In agreement with our data, previous studies reported that overexpression of TRAF6 is sufficient to activate NF-B without additional exogenous stimuli (28), suggesting that overexpression of TRAF6 may cause their oligomerization, which leads to downstream signal events (30, 31).

View larger version (18K): [in this window] [in a new window]   FIGURE 6. TRAF6 requires Spi-1 to activate the IL1B promoter. A TRAF6 expression vector, pcTRAF6 (4 µg), and a Spi-1 expression vector, pECE Spi-1 (4 µg), alone or in combination was cotransfected into EL4 cells along with the IL1B promoter CAT reporter 3MEHT (10 µg) and assayed for TRAF6-induced promoter activity. The total amount of transfected DNA was kept constant (18 µg) by addition of control vector. At 24 h after transfection, the cells were treated with 2 ng/ml IL-1 or left untreated. The results from a representative experiment are shown.

NF-IL6 lacking the transactivation domain (NF-IL6(spl)) inhibits IL-1-induced promoter activity even in the absence of its cognate binding site (3MEHTmNF-IL6)

The IL1B promoter containing an intact Spi-1 site and a mutated NF-IL6 site (HTmNF-IL6) retained significant ability to be activated by IL-1 in THP-1 cells. In the present study, to evaluate functional involvement of NF-IL6 in IL-1 induction ofHTmNF-IL6, various amounts of pcmNF-IL6(spl), encoding a NF-IL6 mutant lacking the transactivation domain, were cotransfected into EL4 cells with a full-length Spi-1 expression vector, pECE Spi-1, and assayed for IL-1-induced promoter activity. The truncated NF-IL6 (NF-IL6(spl)), which lacks amino acids between residues 41 and 205, has been demonstrated to act as a dominant-negative factor in LPS induction of the IL1B enhancer (19). As shown in Fig. 7A, IL-1 induced the promoter containing an intact Spi-1 site and a mutated NF-IL6 site (HTmNF-IL6) in Spi-1-expressing EL4 cells. The IL-1-induced activity for 3MEHTmNF-IL6 was significantly inhibited by expression of the truncated NF-IL6(spl). Cotransfection of 2 µg of pcmNF-IL6(spl) resulted in an 80% loss of IL-1-inducible activity. In contrast, when a full-length NF-IL6 expression vector, pcNF-IL6, was used instead of pcmNF-IL6(spl), cotransfection of pcNF-IL6 enhanced IL-1-induced activity for 3MEHTmNF-IL6 in the presence of Spi-1 (Fig. 7B).

View larger version (21K): [in this window] [in a new window]   FIGURE 7. Inhibition of IL-1-induced CAT activity for 3MEHTmNF-IL6 by expression of a truncated NF-IL6. Increasing amounts of a mutant NF-IL6 expression vector, pcmNF-IL6(spl) (A), or 1 µg of a full-length NF-IL6 expression vector, pcNF-IL6 (B), were cotransfected into EL4 thymoma cells along with 3MEHTmNF-IL6 reporter (10 µg) and pECE Spi-1 (4 µg). The total amount of transfected DNA was kept constant by addition of control vector. At 24 h after transfection, the cells were treated with 2 ng/ml IL-1 or left untreated. The CAT data were normalized to the average activity elicited by the IL-1-induced 3MEHTmNF-IL6 construct in the presence of Spi-1 expression vector cotransfection. Data represent the mean ± SD of three experiments.

IL-1 induces IL-1 mRNA expression in THP-1 monocytes

RT-PCR analysis for IL-1 mRNA expression was performed on total cellular RNA of THP-1 monocytes treated or untreated with IL-1. The cells were carefully maintained in endotoxin-free culture medium. In addition, disposable sterile pipettes and tubes were used to reduce endotoxin levels. As shown in Fig. 8, IL-1 mRNA was detected in IL-1-treated THP-1 cells. In contrast, untreated THP-1 monocytes failed to generate significant signals for IL-1, indicating that IL-1 induces IL1B gene transcription in THP-1 monocytic cells. This result is consistent with our CAT data showing very low levels of background activity in the absence of IL-1.

View larger version (50K): [in this window] [in a new window]   FIGURE 8. IL-1 induces IL-1 mRNA expression in THP-1 monocytic cells. PCR products of IL-1 and -actin were derived from THP-1 cells treated with 10 ng/ml IL-1 for 1 h or untreated. The size of PCR product for IL-1 was 235 bp. Ten microliters of the reaction mixtures were separated at 50 V in 2% agarose gel. Lane 1 shows a m.w. marker.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Spi-1, which is an ETS family member protein restricted in expression to monocytes, macrophages, B lymphocytes, mast cells, and erythroid stem cells, has been shown to be a major determinant in cell type-specific expression of genes encoding CD11b (32) and receptors for GM-CSF (33), M-CSF (34), and G-CSF (35). It was reported that LPS and IFN- induced binding of Spi-1 to the proximal promoter of the MHC class II I-Ab gene in murine tissue macrophages (36). Kim et al. (37) have reported that LPS treatment increases binding of Spi-1 to the IL-18 gene promoter by using RAW 264.7 macrophages. Moreover, we have recently demonstrated that p40 Tax encoded by human T cell leukemia virus-I genome markedly enhances binding of Spi-1 to the IL1B promoter through direct association of Spi-1 with Tax in THP-1 monocytic cells (17). In the present study, EMSA data using IL-1-treated THP-1 cells further showed that IL-1-inducible binding of Spi-1 to the IL1B promoter plays a crucial role in autocrine induction of the IL1B gene in monocytes.

The Spi-1 activation domain comprises at least three functional domains: one that binds TBP, which is necessary for the initiation of transcription; another that contains a Q domain serving as a transactivator; and a third domain that contains the PEST sequence in which phosphorylation of serine 148 is required to bind the lymphoid-specific coactivator NF-EM5/PU.1 interaction partner/IFN regulatory factor-4 (38). In B lymphocytes, Spi-1 promotes binding of NF-EM5/PU.1 interaction partner/IFN regulatory factor-4 to the Ig 3' enhancer (21). The interaction between the two factors contributes to the Ig 3' enhancer activity (38). In contrast, activation of the Ig J chain gene depends upon the amino-terminal portion of Spi-1 (39). The GM-CSFR -chain has been shown to require binding of an uncharacterized factor, PU-SF, to the N-terminal transactivation domain of Spi-1 (33). The Q domain binds CBP/p300 (40). Thus, the functional domains of Spi-1 appear to be differentially used by various genes. In the present study, IL-1-induced Spi-1 activation required the Q and the TBP binding domains (Fig. 5B). The fact that Spi-1 binds adjacent to the IL1B gene TATA box suggests that Spi-1 is involved in the recruitment of TBP to transcription preinitiation complex and in the transcriptional activation through the Q domain.

In addition, it is noteworthy that mutation of a serine to an alanine at codon 148 (S148A) did not affect activation of the IL1B promoter by either IL-1 or PMA. Our transfection studies using neutralizing Abs against IL-1 and IL-1 further showed that activation of the Spi-1 S148A mutant by PMA is not mediated by endogenous IL-1 production (Fig. 5D). Lodie et al. (41) have reported that LPS-induced phosphorylation of Spi-1 at serine 148, located within a casein kinase II motif, increases the capacity of Spi-1 to activate transcription. We also observed that S148A inhibited the ability of Spi-1 to transactivate the IL1B promoter in LPS-treated THP-1 monocytes. In this regard, the fact that LPS can activate the IL1B promoter more strongly than IL-1 and PMA may raise the possibility that, although LPS and IL-1 activate several common genes, LPS-induced phosphorylation of serine 148 recruits an additional transcription factor(s) to activate the IL1B promoter. In contrast, in the Spi-1 domain analysis, Spi-1 proteins were probably expressed in excess of the amount required forreporter vector activation. However, in our EMSA studies using nuclear extracts of EL4 cells transfected with various Spi-1 expression vectors, the relative expression levels of the Spi-1 proteins were almost equal (Fig. 5C). Our transfection data that Spi-1 S148A mutant, unlike wild-type Spi-1 protein, functions as a dominant-negative factor in LPS induction (Fig. 5B) further reveal the importance of serine 148 phosphorylation in LPS induction.

The IL1B promoter contains NF-IL6 binding site, which is located adjacent to the Spi-1 binding motif. NF-IL6, a member of the C/EBP family of leucine zipper transcription factors (42), is abundant in myeloid cells, including THP-1 monocytes. This factor is activated by IL-1 stimulation (42). However, as shown in Fig. 2B, mutation of the -91 to -83 NF-IL6 site (HTmNF-IL6) resulted in only a partial loss of IL-1-inducible promoter activity. The IL-1-induced activity for HTmNF-IL6 was further repressed by expression of a NF-IL6 dominant-negative mutant (Fig. 7A). In this regard, a COOH-terminal ETS winged helix-turn-helix domain of Spi-1 has been demonstrated to be involved in both DNA binding and protein-protein interactions with c-Jun (43), C/EBP proteins (44), human CMV immediate early gene products (45), and other proteins. These results may suggest that in IL-1-stimulated monocytes, NF-IL6 has the capability to activate the IL1B promoter through protein-tethered transactivation mediated by Spi-1. A similar protein-protein interaction between NF-IL6 and Spi-1 was observed in PMA induction (46). In contrast, Shannon et al. (47) have demonstrated that an NF-IL6 binding site in the G-CSF gene promoter is essential for IL-1-induced expression of the G-CSF gene in human fibroblasts. They observed that mutation of a Spi-1 binding site within the G-CSF promoter does not eliminate promoter function in IL-1-treated human fibroblasts. In this regard, the fact that fibroblasts lack Spi-1 expression (48, 49) may explain their results.

In conclusion, we have demonstrated that IL-1 activates its own gene promoter. The IL-1 induction process apparently depends upon IL-1-inducible binding of Spi-1 to its target site within the IL1B promoter. Several transcription factors have been implicated in IL1B gene induction, including CREB, NF-IL6, LPS/IL-1-inducible-STAT, and NF-B (19, 22, 50). An understanding of the mechanism of IL1B gene autoactivation in response to IL-1 is important for future efforts to modulate IL-1-induced pathological processes in various diseases.

	Footnotes

 
¹ Address correspondence and reprint requests to Dr. Junichi Tsukada, First Department of Internal Medicine, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu, 807-8555 Japan. E-mail address: jtsukada{at}med.uoeh-u.ac.jp

² Abbreviations used in this paper: C/EBP, CCAAT/enhancer binding protein; Q, glutamine-rich; CAT, chloramphenicol acetyltransferase; TRAF6, TNFR-associated factor 6; PEST, proline, glutamic acid, serine, and threonine-rich; TBP, TATA-binding protein; IRAK, IL-1R-associated kinase.

Received for publication August 30, 2001. Accepted for publication December 14, 2001.

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