©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning of a Differentially Expressed IB-related Protein (*)

Prabir Ray (§) , Dong-Hong Zhang , Jack A. Elias , Anuradha Ray

From the (1) Department of Internal Medicine, Pulmonary and Critical Care Section, Yale University School of Medicine, New Haven, Connecticut 06520

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have cloned a cDNA corresponding to a novel gene from a human epithelial cell line by subtractive hybridization and polymerase chain reaction techniques. This gene is expressed at the message level and at the protein level in a lung alveolar type II-like epithelial cell line but not in lung fibroblasts. In adult human tissues, the mRNA for this gene was detected only in the heart and the skeletal muscle, but not in the brain, placenta, whole lung, liver, or kidney. We have named this gene IBR (for IB-related) since its 52-kDa protein product has significant homology to the IB family of proteins which function as inhibitory cytoplasmic retention proteins for the vertebrate rel/NF-B transcription factors. Although the important role of NF-B in gene activation in cells of the immune system is now well established, a similar role in other cell types or in vertebrate development is less clear. The deduced amino acid sequence of IBR has the most significant homology to the Drosophila protein Cactus which inhibits the function of the NF-B-like protein Dorsal. In electrophoretic mobility shift experiments, IBR inhibited the ability of the p50:p65 NF-B heterodimer to bind DNA. The DNA binding ability of the p50 homodimer but not the p65 homodimer was drastically inhibited by IBR. In transfection experiments, overexpression of IBR significantly inhibited NF-B-dependent transcription from the Ig enhancer. This new member of the IB family of proteins, IBR, may play an important role in regulation of NF-B function in epithelial cells.


INTRODUCTION

The establishment of specialized cell types during development involves commitment of multipotential stem cells to specific lineages which is associated with expression of cell type-specific genes. It has long been established that morphogenesis of epithelial tissues involves an interaction with the adjacent mesenchyme during embryonic development (reviewed in Ref. 1). Alveolar type II cells are specialized epithelial cells serving important functions in lung development and repair post-injury (2) . To understand the biology of epithelial cell function, we attempted to clone genes that are expressed in type II-like epithelial cells, such as the lung adenocarcinoma cell line A549, but not in lung mesenchymal cells.

We report the cloning of the cDNA for a differentially expressed protein that has significant similarity to IB proteins. Therefore, we have named this protein IBR (for IB-related). Among the IB proteins, overall, Cactus was most similar to IBR. Cactus inhibits the transcription activation function of the NF-B-like protein Dorsal in Drosophila, analogous to the NF-B-IB interaction in mammalian cells (3, 4) . Dissociation of Cactus from Dorsal results in translocation of Dorsal to the nucleus where it activates genes involved in formation of the dorsoventral axis (5, 6) .

NF-B was initially characterized as a heterodimer of a 50-kDa protein (p50) and a 65-kDa protein (p65, now called RelA) that was retained in the cytoplasm by a protein called IB (7, 8, 9) . Activation of NF-B, induced by various agents, including mitogens and cytokines, results in its dissociation from IB, thereby allowing its translocation to the nucleus to activate genes involved in immune functions (10, 11, 12, 13) . It appears that, unlike what was previously thought, phosphorylation of IB may trigger its degradation by cytoplasmic proteases while still complexed to NF-B, rather than cause it to dissociate from NF-B (14) .

IB proteins whose cDNA sequences are known include IB and its avian homolog pp40 (15, 16) , Bcl-3 (17) , the product of the bcl-3 oncogene (18) and IB, a lymphoid-specific form of IB (19, 20) . The sequence of the IB gene is presently unavailable. IB proteins typically contain ankyrin-like repeats, short acidic domains, and consensus sequences for phosphorylation by protein kinase A, protein kinase C, and casein kinase II (7, 8, 9) .

The ankyrin-like repeat sequence is a 33-amino acid motif, first described in the yeast cell cycle control proteins, which consists of a TPLHLA core amino acid sequence and additional well conserved residues (12, 21) . The number and the nature of the conserved amino acids can vary in different proteins. A number of proteins containing ankyrin-like repeats are involved in cell growth and differentiation (12, 21) . It is becoming increasingly apparent that ankyrin repeats mediate protein-protein interactions which are essential in many biological functions, including cellular signaling events (22, 23, 24) .

The cDNA sequence of IBR has an open reading frame of 481 amino acids with three ankyrin-like repeats in its carboxyl terminus. By Western blotting techniques, we identified a 52-kDa protein in epithelial cell lines, but not in fibroblasts using antisera raised against a bacterially expressed fusion protein (glutathione S-transferase linked to the 52-kDa protein). We tested if the result of the data base search showing similarity of the IBR protein with multiple IB-like proteins held up in functional assays. All IB proteins inhibit the binding of specific NF-B protein(s), as demonstrated by electrophoretic mobility shift assays. IBR inhibited the DNA binding ability of an NF-B complex present in nuclear extracts prepared from interleukin-1 (IL-1)() -activated HeLa cells. The binding of a p50 homodimer but not a p65 homodimer was also inhibited by IBR. In transfection experiments, IBR significantly inhibited NF-B-dependent transcription from the Ig enhancer. We postulate that IBR may play an important role in the regulation of NF-B function in epithelial cells.


MATERIALS AND METHODS

Cell Culture All cell lines were obtained from American Type Culture Collection (ATCC). The lung adenocarcinoma cell line A549 was maintained in Dulbecco's modified Eagle's medium:F-12 (1:1) (Life Technologies, Inc.) containing 10% fetal bovine serum (HyClone). The lung fibroblast cells CCL-202 and HeLa cells were maintained in 10% fetal bovine serum-supplemented Dulbecco's modified Eagle's medium containing 1 or 4.5 mg/liter of glucose, respectively. F9 cells were maintained as reported previously (25) . Subtraction, PCR, and Northern Analyses

Synthesis of cDNA and biotinylated Poly(A) RNA

Poly(A) RNA from A549 cells was used in first strand cDNA synthesis (using random hexamers), whereas that from CCL-202 cells was used for preparation of biotinylated RNA as described by Forster et al.(26) .

Enrichment of A549-specific mRNA

To remove the mRNA species common between A549 cells and CCL-202 cells, a 30-fold molar excess of biotinylated mRNA (from CCL-202 cells) over cDNA (from A549 cells) was used in the solution hybridization reaction containing 0.5 M NaCl which was carried out for 24 h at 68 °C. This resulted in a Ro T of 67.5 (mg of RNA/ml of hybridization mix) over 1000 per day which is sufficient for hybridization of even less abundant classes of cellular mRNA sequences which are present in both cell types (26) . cDNA:biotinylated mRNA hybrids were removed with streptavidin followed by phenol extraction and the residual cDNA was recovered by ethanol precipitation and was used as template for PCR amplification. The enrichment step was repeated twice.

PCR Amplification, Cloning, and Sequencing of PCR-amplified Fragment

Two regions in the catalytic domain of type III receptor tyrosine kinases were used to derive oligonucleotide primers for PCR amplification. The sequence of the 5` oligonucleotide primer was 5`-GTCGACAAC/TCTGTTGGGA/GGCCTGC- 3` while that of the 3` primer was 5`-GAATTCAGCAGCCAGGTCTCT/GGTG-3`; the underlined sequences indicate SalI and EcoRI restriction enzyme sites in the 5` and 3` oligonucleotide, respectively. After PCR amplification, the amplified DNA was digested with SalI and EcoRI and cloned into the bacteriophage vector M13mp18. Several recombinant clones were analyzed by sequencing by the dideoxy method (27) .

Northern Analyses

For Northern analyses, 10-µg aliquots of total cellular RNA were denatured with formamide and formaldehyde and then loaded onto 1% agarose gels. The RNA was transferred onto nylon membranes (Micron Separations Inc., Westboro, MA), and hybridization was performed with P-labeled probes following procedures recommended by the manufacturer. Screening of an Epithelial Cell-specific cDNA Library and DNA Sequencing For isolation of the full-length cDNA, a HeLa cell cDNA library in gt11 was screened with the PCR-amplified fragment following standard procedures. The cDNA inserts were subcloned into M13 phage vectors mp18 and mp19 for making deletion constructs and DNA sequencing. A series of nested deletions of the cloned fragments were carried out in an unidirectional (5`-3`) manner using the Cyclone I Biosystem (International Biotechnologies, Inc.). DNA sequencing was performed using the Sequenase kit (U. S. Biochemical Corp.). Both strands were sequenced to obtain an accurate sequence. A composite sequence was derived from overlapping sequences of the progressively deleted fragments. Comparisons with data base sequences were performed using the Blastmail program of the National Center of Biotechnology Information. Construction of a Glutathione S-Transferase-IBR (GSTIBR) Fusion Protein and Preparation of Antiserum The pGEX-2T vector (Pharmacia Biotech Inc.) was used for bacterial expression of an in-frame GSTIBR fusion protein (28) . The recombinant vector was transformed into bacteria deficient in lon protease (strain NB42 of Escherichia coli). Unless otherwise indicated, bacteria were grown at 30 °C in all subsequent steps. A large fraction of the bacterially expressed fusion protein was trapped in inclusion bodies which were recovered following manufacturer's recommendations. For immunization of rabbits, the protein was purified by SDS-PAGE and electroelution.

The fusion protein used in electrophoretic mobility shift assays was purified from inclusion bodies as follows. The insoluble pellet was solubilized in 8 M urea and subsequently dialyzed against 50 mM Tris-HCl, pH 7.5. The renatured protein was purified by affinity chromatography on glutathione-agarose. The protein was eluted by continuous slow stirring of the beads at 4 °C in a buffer containing 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 20 mM reduced glutathione, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride. The eluted protein was dialyzed against 50 mM Tris-HCl, pH 7.5. The purified protein was divided into aliquots and stored at -80 °C. The protein was 98% pure as revealed by SDS-PAGE. The soluble GST control protein was treated with 8 M urea and subjected to similar renaturation steps. The identity of the GST protein and the GSTIBR fusion protein was confirmed by Western blotting using anti-GST antibody. Western Blot Analyses Western blotting was performed by electrophoretic transfer of proteins from SDS-polyacylamide gels onto Immobilon-P (Millipore) membranes. Following incubation of the membrane with primary antibody, the membrane was further processed with alkaline-phosphatase-conjugated mouse anti-rabbit antibody and the detection system of Vector Laboratories. Electrophoretic Mobility Shift Assays Nuclear extracts were prepared from uninduced and IL-1-treated HeLa cells as reported previously (29) . Nuclear extracts or individual proteins were preincubated in the presence or absence of antibodies or competitor proteins for 30 min on ice in a buffer containing 10 mM Hepes, pH 7.9, 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA and 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 2.5 µg/ml poly(dI)poly(dC), and 200 µg/ml bovine serum albumin before addition of the labeled probe and further incubation at room temperature for 15 min. A P-labeled oligonucleotide containing the NF-B sequence from the IL-6 promoter was used as the probe. The binding reactions were analyzed by electrophoresis on 6% native polyacrylamide gels (acrylamide:bisacrylamide = 30:1). Electrophoresis was carried out at 200 V in 0.5 TBE (1 = 0.05 M Tris base, 0.05 M boric acid, and 1.0 mM EDTA) at 4 °C. Gels were dried and subjected to autoradiography. Transfections Transfections in F9 cells were carried out as reported previously (25) . Luciferase assays were performed using the Luciferase Assay System of Promega (Wisconsin) and the luciferase activity was measured in the LB 9501 luminometer of Berthold, Inc. (Bad Wildbad, Federal Republic of Germany).


RESULTS

Subtraction Hybridization and PCR

An enriched population of A549 cell-specific cDNA was obtained by subtractive hybridization with mRNA from lung fibroblast cells. This cDNA was subjected to PCR using degenerate primers from conserved domains present in type III receptor tyrosine kinases which mediate the proliferation and differentiation of many cells by a variety of growth factors (30) . Analysis of the PCR amplification product of A549-specific cDNA by agarose gel electrophoresis revealed amplified DNA fragments in the range from 200 to 600 bp (data not shown).

The DNA sequences of the PCR products were translated in all three reading frames and compared with available sequences in the gene bank. The DNA sequences of all of the PCR-amplified fragments were bound by that of the PCR primers showing primer-specific amplification of cDNAs. The sequences of some of the clones were derived from known receptor tyrosine kinases. The sequence of one clone revealed the presence of an ankyrin-like repeat motif. Since this structural motif is often present in proteins involved in cell cycle control and tissue differentiation, we were persuaded to further characterize this clone. Preliminary analyses indicated that the mRNA specific for this PCR fragment was also expressed in HeLa cells, a cervical carcinoma cell line of epithelial origin. We therefore proceeded to isolate the full-length cDNA from a HeLa cell-derived cDNA library.

cDNA Isolation and Characterization

A HeLa cell cDNA library in the phage vector gt11 was screened with the 276-bp PCR fragment for isolation of the full-length cDNA. This resulted in the isolation of three independent clones. Southern blot analysis at high stringencies showed the presence of a 2-kb insert in one of these clones, a 0.5-kb fragment of which showed specific hybridization to the probe. The other two independent clones contained sequences overlapping with the 2-kb insert.

For sequencing, the DNA inserts from the phage clones were subcloned in both orientations into M13 phage vectors. Sequential deletions of the inserts were obtained for complete sequence information and a composite sequence was derived from the overlapping sequences. The cDNA was found to contain an open reading frame of 481 amino acids (Fig. 1); there are also 324 bp of 5`-untranslated region upstream (data not shown). The ATG codon at nucleotide position 325 is the putative start codon for the full-length gene for the following reasons: ( a) this ATG codon is in the context of a Kozak sequence (31) and the longest open reading frame and ( b) there are stop codons in all reading frames 5` of this ATG codon. Based on the results of the Northern analyses, which showed a 5.5-kb message hybridizing to the probe (Fig. 3), we predict that we are missing 3.5 kb of the 3`-untranslated sequence. Examples of other genes whose transcripts have been shown to contain long untranslated 3`-ends are the vinculin gene (32) and the estrogen receptor gene (33) .


Figure 1: Nucleotide and predicted amino acid sequence of the coding region of the cDNA encoding IBR. The potential protein kinase A, protein kinase C, and casein kinase II phosphorylation sites are indicated by the overlines, the underlines, and the broken lines, respectively. The boxed sequences contain the three ankyrin-like repeat motifs. The termination codon is denoted by an asterisk.




Figure 3: Differential expression of IBR mRNA and protein in epithelial cell lines. A, mRNA expression. Total cytoplasmic RNA was isolated from human tracheal epithelial cell line ( HTE), human alveolar type II-like epithelial cell line ( A549), and a human lung fibroblast cell line ( CCL-202). The RNA (20 µg/lane) was subjected to Northern blot analysis by using the P-labeled PCR fragment (276 bp) as the hybridization probe. RNA molecular size standards are shown on the left in kilobases. Ethidium bromide staining of the same RNAs loaded in a duplicate gel confirmed similar loading of RNA. B, protein expression. HeLa cells or CCL-202 cells were lysed by boiling in SDS-polyacrylamide gel sample loading buffer and were subjected to SDS-PAGE. The fractionated proteins were transferred onto nitrocellulose membrane by electroblotting. Strips of nitrocellulose containing bound protein were incubated with preimmune serum or antiserum from a rabbit immunized with the GST fusion protein. Antibodies bound to proteins were detected by subsequent incubation with alkaline phosphatase-conjugated goat anti-rabbit IgG followed by alkaline phosphatase substrate solution. The positions of protein size markers are indicated on the left. The bottom arrow denotes the expected IBR band. The upper arrow denotes a cross-reacting species of greater molecular size.



The deduced amino acid sequence of the cDNA was compared with available amino acid sequences of other proteins in protein data bases using the BLAST e-mail server. BLAST utilizes an alignment algorithm that identifies similarities without need to introduce gaps and is therefore more stringent (34) . Data base searches revealed that the cDNA we isolated encoded a novel sequence with the following characteristics: ( a) the amino acid sequence between residues 363 and 471 in IBR containing the three ankyrin repeats (Fig. 1) was similar to ankyrin repeats present in IB-like proteins. The overall similarities were as follows: (i) amino acids 363-429 (containing two repeats) and 437-471 (comprising the third repeat) in IBR showed a 32% identity, 49% similarity and a 28% identity, 51% similarity to repeats II-III and repeat IV, respectively, in the Drosophila IB-like protein Cactus (4) ; (ii) amino acids 363-471 in IBR showed a 33% identity, 49% similarity with a stretch containing repeats II, III, and IV present in the precursor of the p50 subunit of NF-B, p105 (12) , and in IB, a lymphoid-specific form of IB whose sequence is identical to the C-terminal part of p105; and last (iv) amino acids 363-424 and 436-464 in IBR showed a 40% identity, 54% similarity and a 34% identity, 58% similarity to repeats II-III and repeat V, respectively, in pp40, the avian homolog of IB (15, 16) . The ankyrin repeats in IBR also showed similarity to those in the erythrocyte protein ankyrin (22) and the G9a protein of presently unknown function (35) . ( b) A 36-amino acid stretch (residues 304-339) rich in glutamate residues showed a 27% identity and 47% similarity with a homologous stretch in the Cactus protein. This region was also found to be similar to a glutamate-rich region in the G9a protein (35) . Fig. 2 illustrates the similarity of IBR with the Drosophila protein Cactus, which overall was most homologous to IBR. Protein motif searches showed the presence of consensus sequences for protein kinase A, protein kinase C, and casein kinase II phosphorylations in IBR which are also commonly found in IB proteins (Fig. 1). It is important to note that no similarity was detected with the Rel homology domain in the Rel family of transcription factors (NF-B), some of which contain ankyrin-like repeats in their precursor forms. It therefore seemed unlikely that the protein encoded by this cDNA was an NF-B-like transcription factor. Again, although the primers for the PCR amplification reaction were derived from receptor tyrosine kinases, the amino acid sequence of IBR did not reveal characteristic motifs present in such receptors. IBR appears to have been a target in the amplification reaction because of coincidental similarity of two regions in the gene, corresponding to the ends of the 276-bp PCR fragment, to the DNA sequences of the PCR primers. In summary, the overall similarity of the IBR protein was greatest with the IB proteins than with other ankyrin-repeat proteins such as members of the Notch family of proteins (36) . Therefore, we named this protein IBR for IB-related.


Figure 2: Summary of the structural features of IBR and sequence comparisons. A, a schematic representation of the primary sequence of IBR and the Cactus protein. The stippled boxes denote the acidic domains in the two proteins, whereas the cross-hatched boxes indicate the ankyrin-like repeats. B, an alignment of the three ankyrin-like repeats in IBR with the general consensus sequence for ankyrin repeats. (C). An alignment of the amino acid sequences of ( a) IBR and Cactus across the ankyrin-like repeats and ( b) across the acidic amino acid-rich regions.



Expression of IBR mRNA and Protein in Epithelial Cells but Not in Fibroblasts

It was expected that the PCR-amplified fragment would be expressed in epithelial cells, but not in fibroblasts, since the template for this reaction was a residue of subtractive hybridization. We investigated by Northern blot analysis if indeed this was true. As illustrated in Fig. 3 A, IBR-specific message of 5.5 kb was detected in A549 cells and in human tracheal epithelial cells. A much smaller transcript of 0.6 kb, insufficient in size to account for the full-length IBR protein, was also detected (this transcript ran off the particular gel shown in Fig. 3 A but is shown in Fig. 4 ). No hybridization was detected with RNA derived from the human lung fibroblast cell line CCL-202. Northern blot analyses therefore indicated that IBR transcripts are expressed in epithelial cells (at least at much higher levels) in preference to fibroblasts. The profile of IBR mRNA expression in other cell lines is presently unknown.


Figure 4: Expression of IBR mRNA in adult human tissues by Northern blot analysis. A human multi-tissue Northern blot (Clontech) was hybridized to the full-length P-labeled IBR cDNA probe. The positions of RNA size markers are shown on the left.



To confirm that the IBR protein was expressed in intact cells, polyclonal antibodies were raised against a bacterial fusion protein consisting of glutathione S-transferase fused to amino acids 39-481 of the IBR protein (GSTIBR). Western blots were prepared from HeLa cells (since these were used to isolate the cDNA) and fibroblast cell lysates. On these blots, immune sera but not preimmune sera detected a band of approximately 52 kDa in lysates prepared from HeLa cells (Fig. 3 B). A fainter second band almost twice in size to this band (approximately 100 kDa in size) was also detected which may be (i) unreduced homodimers or heterodimers of the same protein (mediated by the ankyrin-like repeats) or (ii) a cross-reacting homologous protein. A similar pattern was also identified in proteins recovered from A549 cells (data not shown).

Expression of IBR-specific mRNA in Adult Human Tissues

We investigated the steady state level of IBR-mRNA expression in adult human tissues by Northern blot analysis (Fig. 4). A 5.5-kb transcript, similar in size to that expressed in epithelial cells, was detected only in the heart and the skeletal muscle (Fig. 4). A shorter transcript was detected in all other tissues included in the analysis except for the brain (Fig. 4). This shorter message, insufficient in size to code for a 52-kDa protein, was also detected in epithelial cells and in fibroblasts. The shorter transcript could be an alternately spliced species of the IBR gene. Although this gene is expressed in the alveolar type II-like epithelial cell line A549, one possible reason for not detecting the longer transcript in the lung could be due to the fact that type II epithelial cells constitute only 10% of the adult lung alveolar epithelium. The significance of relatively high expression of the 5.5-kb message in muscle tissue is not presently understood.

IBR Inhibits DNA Binding Activity of NF-B Proteins

To elucidate the biological function of the IBR, the ability of the GSTIBR fusion protein to inhibit the DNA binding activity of NF-B proteins was tested in electrophoretic mobility shift assays. In these experiments, the P-labeled oligonucleotide probe containing an NF-B-site was derived from the IL-6 promoter (25, 37) , and the NF-B protein targets used were (i) the p50:p65 heterodimer present in HeLa cell nuclear extracts, prepared from cells induced with IL-1, a known activator of NF-B (25, 38, 39, 40) , (ii) the p50 homodimer (of the bacterially expressed protein), and (iii) the p65 homodimer (of in vitro translated protein). In electrophoretic mobility shift assays, the DNA binding ability of the p50:p65 heterodimer was inhibited by the GSTIBR fusion protein (Fig. 5 A). The DNA binding activity of the p50 homodimer was significantly inhibited by the fusion protein in a dose-dependent manner (Fig. 5 B). Included in the same experiment, identical concentrations of GSTIBR minimally inhibited the DNA binding activity of the p65 homodimer (Fig. 5 B). The GST polypeptide alone did not affect the DNA binding activity of any of these proteins, indicating that the inhibition was mediated by the IBR part of the fusion protein. The inhibition of DNA binding activity of the p50 homodimer is reminiscent of that of the lymphoid cell-specific IB protein IB (19, 20) . The effect of IBR on the DNA binding ability of c-Rel has not been determined.


Figure 5: The effect of IBR on the binding of NF-B proteins to a target sequence ( A and B) and suppression of p50/p65-mediated activation of the Ig enhancer in F9 cells ( C). A, nuclear extracts from uninduced and IL-1-treated HeLa cells were incubated with P-labeled oligonucleotide containing the NF-B sequence from the IL-6 promoter in the presence or absence of antibodies to the different subunits of NF-B and to the c-Fos protein (as a negative control) or in the presence and absence of 25 or 12.5 ng of GSTIBR or equimolar amounts of GST as competitors. B, the DNA binding reaction was carried out with purified p50 protein (Promega) or in vitro translated p65 in the presence or absence of antibodies to the proteins, as indicated or in the presence of 25, 12.5, or 5 ng of GSTIBR or equimolar amounts of GST. All other conditions were as in A. The binding reactions were analyzed by electrophoresis on 6% native polyacrylamide gels. Gels were dried and subjected to autoradiography. C, effect of IBR on NF-B-dependent transcription. F9 cells were transfected with 5 µg of the reporter plasmid pBIIXLUC containing two B sites from the Ig enhancer driving the expression of a luciferase reporter gene together with equimolar concentrations (1 µg each) of expression plasmids for the p50 and the p65 subunits of NF-B, 2 µg of expression vector for IBR expressing the IBR-mRNA in the sense ( S) or antisense ( AS) orientation and 2 µg of RSVgal (a constitutive expression vector for -galactosidase for monitoring transfection efficiency). 24 h after transfection, cells were harvested and assayed for -galactosidase and luciferase activity. The absolute basal luciferase activity was, on average, 3500 relative light units in these experiments. This is an average of three independent experiments with deviations of no more than 10% between experiments.



Inhibition of NF-B-mediated Activation of the Ig Enhancer by IBR

We also examined the effect of IBR on NF-B-mediated activation of the Ig enhancer in F9 (a murine embryonal carcinoma cell line) cells which lack endogenous Rel-like activity (41) . F9 cells were transiently transfected with a plasmid construct that contained two B sites from the Ig enhancer linked to a luciferase reporter gene together with expression vectors for the p50 and the p65 subunits of NF-B. A combination of p50 and p65 efficiently activated expression from the B sites in the reporter construct (Fig. 5 C). When the p50 and p65 expression vectors were cotransfected with an expression vector for IBR, expression of the reporter gene was significantly inhibited (Fig. 5 C). However, no such suppression was observed when a plasmid construct expressing antisense ( AS) IBR was cotransfected with the NF-B subunits (Fig. 5 C). The expression of the IBR expression vector containing the IBR cDNA in either orientation (sense or antisense) by themselves had no effect on expression of the reporter gene in the absence of the NF-B subunits (Fig. 5 C). Based on all the experiments in Fig. 5, it is too early to decide which one of the mechanisms, inhibition of DNA binding activity of NF-B or inhibition of nuclear translocation of NF-B or both, are relevant to IBR activity in vivo. It is also not known if the in vivo target of IBR is p50. However, the data illustrated in Fig. 5indicate that IBR has IB-like functions.


DISCUSSION

We report the cloning and characterization of the cDNA for a molecule which is differentially expressed in epithelial cell types but not in fibroblasts and is related to the IB family of proteins. The 5.5-kb mRNA corresponding to this newly identified gene encodes a protein product of 481 amino acids with a predicted molecular mass of 52 kDa. Intracellular expression of a protein of expected size was confirmed using antisera raised against a GSTIBR fusion protein.

Among the presently characterized IB proteins, overall, the amino acid sequence of the Drosophila Cactus protein was most similar to that of IBR. It is intriguing that while most IB proteins, including Cactus, contain five to seven ankyrin repeats, this newly identified member of the family contains only three repeats. Another recently identified gene ikbl (for I kappa B-like) encodes a protein product containing two and a half ankyrin repeats, which bear marked similarity to those present in the IB family of proteins (42) . The difference in the repeat numbers between IBR and the other IB proteins may reflect differences in the nature of the interacting partners of the two classes of proteins. This may have implications in the normal physiological function of IBR. It might be expected that the IB proteins with fewer repeats such as IBR and Ikbl interact with a different set of NF-B/Rel proteins which respond to distinct stimuli.

The ankyrin repeats typically contain a core amino acid sequence (TPLH) and sequences outside of this core are extremely variable. Different repeats within a given IB molecule are more variable than individual repeats between members of the same class. In ankyrin, the different repeats have different binding activities (43) . It has been hypothesized that the repeats in their folded conformation expose variable residues that are capable of interacting with distinct protein partners. By this line of reasoning, the fact that the ankyrin repeats in IBR showed the highest homology to those in Cactus and other members of the IB family suggested that IBR might have IB-like functions.

In keeping with this argument, our data indicate that IBR can inhibit the DNA binding ability of NF-B. This is similar to the properties of most IB proteins which upon interaction with a Rel complex inhibit the DNA binding ability of the Rel complex. Transfection experiments clearly indicate that IBR can inhibit activation of NF-B sites mediated by a combination of p50 and p65, as might be expected for IB proteins. However, the precise mechanism of this inhibition is unknown. The inhibition could potentially be due to retention of the p50:p65 complex in the cytoplasm or due to the ability of IBR to remove preformed NF-B complexes on DNA, as was demonstrated for IB and Bcl-3 in in vitro studies (44) . Each IB protein usually has a unique spectrum of activity for inhibiting DNA binding by Rel homodimers and heterodimers. The true physiological partner(s) of IBR remains to be identified. Differential expression of IBR in epithelial cells raises a question whether IBR is involved in special functions of these cell types. For example, is IBR involved in integrating proliferating/differentiating signals that are called into action during re-epithelialization of the lung following injury? Identification of the physiological partner(s) of IBR will help elucidate the physiological function of IBR.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant HL 52014, a grant from the American Lung Association (to P. R.), and National Institutes of Health Grant AI 31137 (to A. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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 GenBank/EMBL Data Bank with accession number(s) U16258.

§
To whom correspondence should be addressed. Tel.: 203-785-3620; Fax: 203-785-3826.

The abbreviations used are: IL, interleukin; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; bp, base pair(s); kb, kilobase(s).


ACKNOWLEDGEMENTS

We thank Gary Nabel for providing us with plasmid constructs containing the p65 cDNA and Sankar Ghosh for the p50 expression vector and pBIIXLUC. We thank Lynna Stone-Infeld for assistance with data base searches and Mandy Abkowicz for technical assistance. We thank members of the Pulmonary and Critical Care Section for numerous helpful discussions.


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