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Isolation of a Novel Interleukin-1-inducible Nuclear Protein Bearing Ankyrin-repeat Motifs*

Hirotaka Haruta, Akira KatoDagger, and Kazuo Todokoro§

From the Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), Koyadai 3-1, Tsukuba, Ibaraki 305-0074, Japan

Received for publication, February 7, 2001, and in revised form, February 24, 2001



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We isolated a novel gene termed interleukin (IL)-1-inducible nuclear ankyrin-repeat protein (INAP), of which expression was specifically induced by IL-1 in OP9 stromal cells. The INAP has ankyrin-repeat motifs and shares weak amino acid sequence homology with Bcl-3 and other Ikappa B family members. The human genomic INAP gene found in the NCBI data base is located at chromosome 3q3.11. Northern blot analyses revealed that INAP was not expressed in any examined tissues without stimulation, but INAP expression was rapidly and transiently induced by IL-1 although not by tumor necrosis factor alpha  nor by phorbol 12-myristate 13-acetate in OP9 cells. Immunoblots with anti-INAP-specific antibody demonstrated that INAP was rapidly and specifically produced by IL-1 stimulation and was predominantly localized in the nucleus. Immunofluorescence stainings showed that the INAP newly synthesized by IL-1 stimulation was promptly translocated into the nucleus, and FLAG-tagged INAP forcibly expressed in NIH/3T3 cells was also specifically localized in the nucleus. The possible interaction of INAP with RelA/p65, NF-kappa B1/p50, NF-kappa B2/p52, C/EBPbeta , and retinoid X receptor was examined, but we could detect none of these interactions in the nuclear extracts of IL-1-stimulated cells. Unlike Bcl-3 and other Ikappa B family members, INAP may play a unique role in IL-1-induced specific gene expression and/or signal transduction in the nucleus.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

NF-kappa B is a transcription factor that is known to play an important role in regulating immune and inflammatory responses (1-3). There are presently five members of the mammalian NF-kappa B/Rel family, NF-kappa B1/p50, NF-kappa B2/p52, c-Rel, RelA/p65, and RelB (1-3). The classic form of NF-kappa B, the heterodimer of the NF-kappa B1/p50 and RelA/p65, is normally retained in the cytoplasm through interactions with inhibitor protein Ikappa B. The Ikappa B family of proteins includes Ikappa Balpha , Ikappa Bbeta , Ikappa Bepsilon , Bcl-3, NF-kappa B1/p105, and NF-kappa B2/p100, all of which possess 5-7 ankyrin-repeat motifs (1-3), which form a functional unit able to interact with the Rel homology domain of NF-kappa B. The cytoplasmic retention of the classic form of NF-kappa B is primarily carried out by Ikappa Balpha and Ikappa Bbeta (4-7). Inductive stimuli, such as tumor necrosis factor alpha  (TNFalpha ),1 interleukin-1 (IL-1), and bacterial endotoxin, lead to the phosphorylation and degradation of Ikappa B, allowing NF-kappa B to translocate into the nucleus and regulate specific gene expression (1-3).

Ikappa Balpha is degraded in response to the NF-kappa B inducers TNFalpha , IL-1, lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), and double-stranded RNA. In contrast, Ikappa Bbeta is degraded only when cells are stimulated with IL-1 or LPS, both of which cause persistent long term activation of NF-kappa B (4-7). Following degradation of the initial pool of Ikappa Bbeta in response to IL-1 or LPS, newly synthesized Ikappa Bbeta accumulates as an unphosphorylated protein that forms a stable complex with NF-kappa B and prevents it from binding to newly synthesized Ikappa Balpha (4-7), resulting in the prolonged activation of NF-kappa B (4, 8). This unphosphorylated Ikappa Bbeta cannot block the nuclear localization signal of NF-kappa B, thus this NF-kappa B·Ikappa Bbeta complex translocates into the nucleus. The function of this complex in the nucleus is yet to be elucidated, and the mechanism by which only IL-1 and LPS can degrade Ikappa Bbeta remains to be resolved.

Unlike the other Ikappa B family members, Bcl-3 is a nuclear protein (9-11). It does not bind to RelA/p65 but specifically binds to NF-kappa B1/p50 or NF-kappa B2/p52 homodimers (10, 12-14) and takes them into the nucleus where it exhibits transactivating activity (11, 15). The formation of Bcl-3·(NF-kappa B1/p50)2·kappa B complex or Bcl-3·(NF-kappa B2/p52)2·kappa B complex is regulated by the phosphorylation status of Bcl-3 (14, 16). Bcl-3 also interacts with retinoid X receptor (RXR) or activating protein-1 (AP-1) and functions as their transcription coactivator (17, 18). However, the detailed characters of this unique Ikappa B family member remain mysterious.

Here we identified a novel Ikappa B family member, termed IL-1-inducible nuclear ankyrin-repeat protein (INAP), of which expression is specifically induced by IL-1. INAP was found to be weakly homologous to Bcl-3 and localized in the nucleus like Bcl-3. We discuss here the possible function of this novel Ikappa B family member.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Isolation of INAP cDNA-- OP9 cells were cultured in alpha -minimum Eagle's medium supplemented with 20% fetal calf serum (FCS) with or without 10 ng/ml of mouse IL-1alpha (Genzyme/Techne) in the presence of 10 µg/ml of cycloheximide for 1 h, and total mRNA was isolated. The PCR-Select cDNA subtraction kit (CLONTECH) was used for cDNA synthesis and suppressive subtractive hybridization, according to the manufacturer's instructions. The cDNA from OP9 cells incubated with IL-1alpha was used as the tester sample, and that from untreated cells was used as the driver sample. The 5' end of INAP cDNA was confirmed by the rapid amplification of the cDNA ends (5' RACE) method.

Northern Blot Analysis-- Total mRNAs from OP9 cells stimulated with 10 ng/ml of mouse IL-1alpha , 20 ng/ml of mouse TNFalpha (Genzyme/Techne), or 100 ng/ml of PMA (Sigma) for the indicated period of time were isolated. Total mRNA (20 µg/lane) was resolved on a 1% agarose gel and transferred to a Hybond-N+ membrane. The filters were hybridized with the digoxigenin (DIG)-labeled INAP cDNA probe (nucleotides 838-1368) at 42 °C for 16 h in DIG Easy Hyb solution (Roche Diagnostics). After washing at 68 °C for 30 min in 1 × SSC containing 0.2% SDS, the hybridized bands were detected by chemiluminescent detection using CDP-StarTM substrate (Roche Diagnostics).

Preparation of Glutathione S-Transferase (GST)-INAP and Anti-INAP Antibody-- The INAP cDNA (amino acids 108 to 403) was ligated into pGEX2T and expressed in Escherichia coli BL21(DE3) pLysS cells. Rabbit antiserum was raised against GST-INAP, and the polyclonal antibody was purified by GST-INAP affinity chromatography.

Indirect Immunofluorescence Staining of OP9 Cells-- Cells on coverslips were fixed with 3% formaldehyde and 0.2% Triton X-100 for 15 min. The cells were blocked with 5% FCS in PBS and incubated with purified anti-INAP-specific rabbit antibody. The cells were then reacted with Cy3-conjugated F(ab')2 fragment donkey anti-rabbit antibody (Jackson ImmunoResearch Laboratories) and 1 mg/ml 4,6-diamidino-2-phenylindole (DAPI). The coverslips were mounted in PBS containing 90% glycerol and 0.1% 1,4-para-phenylene diamine and were observed under a fluorescence microscope (Olympus BX60-34-FLBD1).

Immunoblot Analysis-- Cells were sonicated in 20 mM Hepes, pH 7.4, 1 mM EDTA, 0.1 mM EGTA, 2 mM MgCl2, 1 mM Na3VO4, 20 mM NaF, 150 mM NaCl, 5% glycerol, 0.2% Nonidet P-40, 1 µg/ml pepstatin A, 1 µg/ml aprotinin, 5 µg/ml leupeptin, and 5 µg/ml Pefabloc SC. Samples were fractionated by 10% SDS-PAGE and electrotransferred to an ECL membrane. The membrane was blocked with 5% milk in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.5% Tween 20, incubated with anti-INAP-specific rabbit antibody for 2 h, and then incubated with horseradish peroxidase-conjugated F(ab')2 fragment donkey anti-rabbit antibody. The antibody complexes were visualized by an ECL system (Amersham Pharmacia Biotech).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Isolation and Structure of INAP-- We attempted to isolate the genes of which transcriptions were induced by IL-1 in mouse stromal OP9 cells. Subtraction of mRNAs of OP9 cells before and after IL-1 stimulation led to the isolation of a number of cDNA fragments encoding factors related to the IL-1 response. Among them, we focused on a novel gene, termed INAP. The isolated mouse full-length INAP cDNA (2339 bp) contained an open reading frame, which encoded a polypeptide of 728 amino acids with a calculated molecular mass of 79,007 daltons and a predicted pI of 6.45. A search in the NCBI data base using the BLAST program revealed that a human genomic sequence (map element NT_022504) of chromosome 3 contained the human INAP gene, which consists of 12 exons and is located at chromosome 3q13.11. Human INAP constituted of 718 amino acids has a predicted molecular mass of 78,061 daltons. Human INAP has 82% amino acid identity and 85% similarity with mouse INAP and has a 10-amino acid deletion at amino acid 301-310 compared with mouse sequences.

Fig. 1A shows the schematic drawings of the isolated mouse INAP cDNA and of the human genomic INAP gene found in the NCBI data base. Mouse and human INAP were found to be weakly homologous to the Ikappa B family and the Rel family. The most striking feature of mouse and human INAP is that both INAP contain five highly conserved ankyrin-repeat motifs in carboxyl-terminal regions. Using PESTFIND software, it was also found that both INAP have PEST (P, E, D, S, and T residue-rich)-like sequences, which are implicated in the rapid turnover of proteins (19). Although Ikappa Balpha and Ikappa Bbeta have PEST sequences in carboxyl-terminal ends, both mouse and human INAP have them at amino-terminal regions (amino acids 11-84 and 185-203 in mouse and 184-204 in human). A serine-rich region was also found in amino-terminal regions (amino acids 53-77 in mouse and 51-86 in human), and a glutamine-rich region was found in the middle regions (amino acids 245-308 in mouse and 247-299 in human). However, the glycine-rich region, Rel homology domain, or obvious nuclear localization signal, which commonly exist in the Rel family, was not found in INAP. Reinhardt's method (20) for cytoplasmic or nuclear discrimination predicted that there is a 94% possibility that INAP is localized in the nucleus.


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Fig. 1.   Structure of INAP. A, schematic drawings of mouse full-length INAP cDNA (mINAP) and of the human genomic INAP gene (hINAP). The locations of the serine-rich region, glutamine-rich region, PEST-like sequences, and ankyrin-repeat motifs are indicated. B, phylogenetic tree of INAP and its related proteins. The phylogenetic tree was constructed by the neighbor-joining method using ClustalX 1.8 based on the alignment and visualized with the Treeview program 1.6.1. Species abbreviations are as follows: h, Homo sapiens; m, Mus musculus; r, Rattus norvegicus; b, Bos taurus; and s, Sus scrofa.

To examine the relationship of INAP to other members of the Ikappa B and Rel families, a phylogenetic tree was constructed using the amino acid sequences of all known mammalian Ikappa B and Rel families (Fig. 1B). The closest protein of INAP turned out to be Bcl-3, and the second closest was Ikappa Bepsilon . Mouse INAP has 30-33% identity and 36-38% similarity with human Bcl-3 and mouse Bcl-3. Mouse INAP has 26-29% identity and 33-36% similarity with those of the other Ikappa B family members and has 22-35% identity and 22-41% similarity with those of Rel family members.

INAP Expression Is Rapidly and Specifically Induced by IL-1 Stimulation-- Expression of INAP in various mouse tissues was examined by Northern blot analyses, but no INAP mRNA was detectable in any tissues examined because of its sparse expression (data not shown). Therefore, we examined INAP expression in OP9 cells at various time points after IL-1alpha stimulation (Fig. 2, right panel). Although no INAP mRNA was detected in unstimulated OP9 cells (Fig. 2, right panel, lane 1), a single hybridized band was weakly detected 15 min after IL-1alpha stimulation (lane 2). The level of INAP mRNA increased and reached the maximum at 1 h after IL-1alpha stimulation (lane 4) and then decreased thereafter (lanes 5, 6), indicating that the INAP gene was rapidly and transiently transcribed after IL-1alpha stimulation in OP9 cells. IL-1beta also exhibited the same effect on INAP expression (data not shown). Similarly, INAP expression after TNFalpha or PMA stimulation was examined, but no transcript was detected (Fig. 2, left and middle panels) although OP9 cells are responsive to TNFalpha and PMA, indicating that INAP transcription was specifically induced by IL-1 stimulation.


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Fig. 2.   INAP transcripts were specifically induced by IL-1. Northern blot analysis of INAP mRNA expression in OP9 cells is shown. OP9 cells were treated with TNFalpha (left panel), PMA (middle panel), or IL-1alpha (right panel) for the indicated time period, and total mRNA (20 µg/lane) blotted on the filters was hybridized with DIG-labeled INAP cDNA probe.

The expression of mouse INAP in the protein level was examined by immunoblot analyses with a purified anti-INAP-specific rabbit antibody. As shown in Fig. 3A, mouse INAP of 79 and 82 kDa were clearly detected in IL-1alpha (right panel)- but not TNFalpha (left panel)- or PMA (middle panel)-stimulated OP9 cells, confirming that INAP expression was specifically induced by IL-1alpha . The double bands were clearly detected within 30 min after IL-1alpha stimulation (Fig. 3A, right panel, lane 2), although they were not seen in the cells without stimulation (lane 1). Although INAP transcripts were transiently expressed, the protein level increased until 1 h after IL-1alpha stimulation and retained its level even 24 h after stimulation (lanes 3-5), suggesting that the newly synthesized INAP is relatively stable and accumulates in the cells. We also detected human INAP in IL-1alpha -stimulated HeLa cell extracts by the same antibody (data not shown).


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Fig. 3.   Immunoblot analyses of INAP. A, INAP protein was specifically produced by IL-1 stimulation. OP9 cells were treated with TNFalpha (left panel), PMA (middle panel), or IL-1alpha (right panel) for the indicated time period, and total cell extract (100 µg/lane) blotted on the filters was reacted with purified anti-INAP-specific rabbit antibody. Arrows indicate double bands of INAP. Location and size (in kDa) of protein standards are shown at the left. B, INAP was not phosphorylated and was localized in the nucleus. OP9 cells were treated with (+) or without (-) IL-1, and nuclear extracts and cytosolic extracts were prepared. The INAP was immunoprecipitated with purified anti-INAP-specific rabbit antibody, treated (+) or untreated (-) with CIP, and separated by SDS-PAGE. The blotted proteins were reacted with anti-INAP rabbit antibody. Arrows indicate double bands of INAP.

The double bands were clearly recognized by anti-INAP antibody, and thus we speculated that the upper band might be the phosphorylated form of the lower band. The INAP was therefore immunoprecipitated with its specific antibody from the nuclear extracts, as well as the cytosolic extracts, which were prepared from OP9 cells treated with or without IL-1alpha . The immunoprecipitates were treated with calf intestine alkaline phosphatase (CIP), separated by SDS-PAGE, and immunoblotted with anti-INAP antibody. The results demonstrated that INAP was predominantly detected in the nuclear extracts and that the double bands were not affected by phosphatase treatment (Fig. 3B), indicating that INAP was not phosphorylated and that subcellular localization of INAP was not affected by its phosphorylation status. We therefore concluded that the upper band was not the phosphorylated form of the lower band. We further noticed that the INAP has a second Met codon at amino acid 26, and thus the lower band may be the protein product translated from this second Met codon.

INAP Is Rapidly Translocated into the Nucleus-- Subcellular localization of INAP in OP9 cells with or without IL-1alpha stimulation for 1 h was examined by indirect immunofluorescence microscopic analysis, and the fluorescent images were overlaid on difference interference contrast images (Fig. 4A). The INAP stained with purified anti-INAP-specific antibody in red were predominantly detected in the nuclei of OP9 cells treated with IL-1alpha for 1 h (Fig. 4A, left lower panel), whereas it was rarely seen in the cells prior to IL-1alpha stimulation (left upper panel). The chromosomes stained with DAPI in blue (right lower panel) were completely overlapped with INAP stained in red (left lower panel). These results clearly demonstrated that newly synthesized INAP was promptly translocated into the nucleus by IL-1alpha stimulation in OP9 cells. Furthermore, we found that IL-1 stimulated the production of INAP in various mouse organs including spleen, small intestine, lung, liver, heart, and kidney and that INAP was always localized in nucleus in these IL-1-stimulated tissues (data not shown). Thus, IL-1-specific INAP expression and its nuclear localization are not specific events observed only in OP9 cells.


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Fig. 4.   Newly synthesized INAP was translocated into the nucleus. A, OP9 cells stimulated (lower panels) or unstimulated (upper panels) with IL-1alpha for 1 h were stained with anti-INAP-specific rabbit antibody followed by Cy3-conjugated F(ab')2 fragment donkey anti-rabbit antibody (left panels). The nuclei (chromosomes) of the same cells were also stained with DAPI (right panels). Fluorescent images are overlaid on difference interference contrast images. The INAP in the nucleus is shown in red, and chromosomes are shown in blue. B, NIH/3T3 cells expressing FLAG-tagged INAP were stained with anti-FLAG mouse monoclonal antibody followed by fluorescein isothiocyanate-labeled F(ab')2 fragment goat anti-mouse antibody. The FLAG-INAP in the nucleus is shown in green, and chromosomes are shown in blue.

To confirm these findings, FLAG-tagged INAP and HA-tagged INAP were transiently expressed in mouse fibroblast NIH/3T3 cells. Fig. 4B shows that FLAG-tagged INAP was clearly detected only in the nucleus of the transfected cells by anti-FLAG antibody (stained in green; left panel). The chromosomes stained with DAPI in blue (right panel) were completely overlapped with INAP stained in green (left panel) in transfectants. Similarly, HA-tagged INAP was also localized in the nucleus (data not shown). Taken together, these results clearly indicate that INAP was promptly translocated into the nucleus after INAP protein synthesis was induced by IL-1 stimulation.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We isolated a novel IL-1-inducible nuclear factor, INAP, which is related to the Ikappa B family and the Rel family. It is well known that Ikappa B family members bind to the RelA/p65·NF-kappa B1/p50 complex and prevents the complex from activating and translocating into the nucleus. Therefore, possible interaction of INAP with the RelA/p65·NF-kappa B1/p50 complex was examined by immunoprecipitation followed by immunoblot analysis. However, we failed to detect the direct and/or indirect binding of INAP to RelA/p65 (data not shown). Furthermore, one of the most important factors induced by IL-1 stimulation is IL6, of which gene expression is regulated by C/EBPbeta (NF-IL6), AP-1, and NF-kappa B (21, 22). Therefore, we also examined the possible interactions of INAP with C/EBPbeta and c-Fos/c-Jun in nuclear extracts prepared from IL-1alpha -stimulated OP9 cells. Once again, we could not detect the interactions of INAP with C/EBPbeta or with c-Fos/c-Jun (data not shown). Moreover, the fact that Bcl-3, the protein most closely related to INAP, associates NF-kappa B1/p50 or NF-kappa B2/p52 homodimers and modulates their transactivation activities (11, 16) motivated us to examine whether INAP associates with NF-kappa B1/p50 or NF-kappa B2/p52 in IL-1-stimulated nucleus. None of these interactions, however, was detected by immunoprecipitation followed by immunoblot analysis (data not shown). It has also been reported that Bcl-3 binds to RXR (17) or to AP-1 (18) and regulate specific gene expression, and thus we also examined the possible interactions of INAP with RXR or with AP-1 but failed to detect the bindings (data not shown). We concluded that INAP could not bind to any of the binding partners with which Bcl-3 has been reported to interact and that INAP is a very unique protein in the Ikappa B family and is clearly distinct even from the most closely related Ikappa B family member, Bcl-3. To determine the biological function of INAP on IL-1 signalings it is very important to identify the INAP-binding proteins in the IL-1-stimulated nucleus by other means such as yeast two-hybrid screening, pull-down experiments, and far-Western screening.

INAP was found to be a novel nuclear factor related to the Ikappa B family, but by IL-1 stimulation INAP was newly produced and accumulated in the nucleus, rather than being degraded as were other Ikappa B family members. From a gene expression point of view, INAP is quite a distinct protein from these family members. There exists no obvious nuclear localization signal in INAP, but it does exist in the nucleus. Furthermore, INAP was not phosphorylated no matter whether it was localized in the cytoplasm or nucleus, indicating that the phosphorylation status of INAP does not affect its subcellular localization.

A few potential NF-kappa B binding sites were found around -340 bp upstream from the initiation codon in human INAP gene promoter regions. However, we demonstrated that INAP gene expression was rapidly induced by IL-1 but not by TNFalpha nor by PMA, all of which are known to activate the NF-kappa B signaling pathway. Thus, INAP gene expression is not simply regulated by NF-kappa B signaling. IL-1- and LPS-specific persistent activation of NF-kappa B has been reported (4, 5), but rapid IL-1- (and LPS-) specific INAP gene expression cannot be explained by this mechanism. The mechanism is thus obscure at this moment, and further analyses are required.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) AB026551.

Dagger Present address: Dept. of Biological Sciences, Tokyo Inst. of Technology, Yokohama, Japan.

§ To whom correspondence should be addressed. Tel.: 81-298-36-9075; Fax: 81-298-36-9090; E-mail: todokoro@rtc.riken.go.jp.

Published, JBC Papers in Press, March 2, 2001, DOI 10.1074/jbc.C100075200

    ABBREVIATIONS

The abbreviations used are: TNFalpha , tumor necrosis factor alpha ; IL-1, interleukin-1; PMA, phorbol 12-myristate 13-acetate; DAPI, 4,6-diamidino-2-phenylindole; LPS, lipopolysaccharide; RXR, retinoid X receptor; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; AP-1, activating protein-1; INAP, IL-1-inducible nuclear ankyrin-repeat protein; FCS, fetal calf serum; DIG, digoxigenin; ECL, enhanced chemiluminescence; bp, base pair; CIP, calf intestine alkaline phosphatase; HA, hemagglutinin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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