Interferon gamma -dependent Induction of Human Intercellular Adhesion Molecule-1 Gene Expression Involves Activation of a Distinct STAT Protein Complex*

(Received for publication, February 27, 1996, and in revised form, September 10, 1996)

Shubhada M. Naik , Naotaka Shibagaki Dagger , Lian-Jie Li §, Kimberly L. Quinlan , Lani L. L. Paxton and S. Wright Caughman

From the Emory Skin Diseases Research Core Center, Department of Dermatology, Emory University School of Medicine, Atlanta, Georgia 30322

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

In response to interferon gamma  (IFNgamma ), intercellular adhesion molecule-1 (ICAM-1) is expressed on human keratinocytes, a cell type that is critically involved in cutaneous inflammation. An ICAM-1 5' regulatory region palindromic response element, pIgamma RE, has been shown to confer IFNgamma -dependent transcription enhancement. By electrophoretic mobility shift assays (EMSA), pIgamma RE forms a distinct complex with proteins from IFNgamma -treated human keratinocytes, termed gamma  response factor (GRF). Binding of GRF is tyrosine phosphorylation-dependent, and mutations of pIgamma RE that disrupt the palindromic sequence or alter its spatial relationship abrogate GRF binding. Supershift EMSAs using antibodies to characterized STAT proteins suggest that GRF contains a Stat1alpha -like protein; however, non-ICAM-1 IFNgamma -responsive elements (REs) known to bind Stat1alpha homodimers fail to compete for GRF binding in EMSA, and pIgamma RE does not cross-compete with these REs that complex with homodimeric stat1alpha . The pIgamma RE·GRF complex also displays a distinctly different electrophoretic mobility compared to that of IFNgamma REs complexed to homodimeric Stat1alpha . These findings indicate that a distinct complex containing a Stat1alpha -like protein mediates IFNgamma -induced ICAM-1 gene transcription and identifies a subset of IFNgamma -responsive genes that appear to be regulated by this complex.


INTRODUCTION

The initiation and evolution of localized inflammation is a consequence of homing and extravasation of leukocytes at sites of tissue injury or threat (1). Intercellular adhesion molecule-1 (ICAM-1)1, a cell surface glycoprotein that belongs to the immunoglobulin gene superfamily, serves as a specific ligand for receptors expressed by leukocytes (2). ICAM-1 plays a pivotal role in the adhesion and transmigration of leukocytes at sites of inflammation, and the up-regulation of ICAM-1 cell surface expression during inflammatory responses is essential in facilitating leukocyte migration (3, 4).

IFNgamma , a pleiotropic cytokine produced by activated T lymphocytes, plays a critical role in host defenses and inflammation (5). In the skin IFNgamma induces de novo expression of ICAM-1 on human keratinocytes (HK), cells that are critically involved in cutaneous inflammatory processes (6). Upon binding to its receptor, IFNgamma initiates a signal transduction cascade that involves rapid activation of two members of the Janus tyrosine kinase family, JAK-1 and JAK-2 (7), and consequent tyrosine phosphorylation and activation of a latent cytoplasmic protein, originally referred to as p91 and now known as Stat1alpha (8, 9). Stat1alpha is the first described member of a family of proteins known as STATs, or signal transducers and activators of transcription. When activated by IFNgamma , activated Stat1alpha homodimerizes through Src homology domains (10, 11) to form gamma -activated factor (GAF). The Stat1alpha homodimers, after translocation to the nucleus, bind to the gamma -activated site (GAS), first identified in the human guanylate-binding protein (GBP) gene, and initiate gene transcription (8, 9, 12). Stat1beta (p84) is an alternatively spliced product of the gene for Stat1 and is a truncated protein that lacks 38 amino acids at the carboxyl end of Stat1. Stat1beta also homodimerizes and is capable of binding GAS but does not activate transcription (13). It has recently been shown that Stat1alpha , which obligatorily requires tyrosine phosphorylation to become active, also requires phosphorylation of a serine residue for maximal activation of gene transcription (14).

In addition to formation of the homodimeric GAF, Stat1 also participates in forming the heteromultimeric transcription complex ISGF3, composed of Stat1alpha , stat2, and a non-STAT protein, p48 (15, 16, 17, 18). ISGF3 binds to the IFN-stimulated response element (ISRE), which is an IFNalpha /beta - and IFNgamma -inducible element involved in the regulation of a variety of genes (11, 13, 17). Recently, multiple cytokines and growth factors have been shown to mediate their transcriptional effects through these and additional STAT proteins, of which there are now six characterized members (19). However, Stat1alpha is frequently activated in response to a wide variety of extracellular signals, including those involved in transcriptional activation of a number of genes involved in immune responses (19).

IFNgamma -dependent induction of ICAM-1 gene expression is regulated at the transcriptional level (20, 21). The 5' flanking region of ICAM-1 gene contains an 11-base pair (bp) element, which we refer to as palindromic IFNgamma response element (RE), or pIgamma RE, located upstream of the ICAM-1 transcription initiation site between nucleotides -76 and -66. pIgamma RE is composed of the sequence 5'-TTTCCGGGAAA-3'. Several laboratories have demonstrated that pIgamma RE is both necessary and sufficient for IFNgamma -dependent gene transcription (20, 22).2

The present studies were designed to characterize the molecular events and trans-acting factors involved in the IFNgamma -induced regulation of ICAM-1 gene transcription. The data presented show that the protein complex activated by IFNgamma , which trans-activates ICAM-1 gene expression by binding to pIgamma RE, shares both similarities and distinct differences with previously characterized IFNgamma -activated STAT complexes. From these data we propose that the protein complex mediating IFNgamma -dependent ICAM-1 gene transcription, which we refer to as the gamma  response factor, or GRF, represents a distinct form of IFNgamma -induced transcription trans-activator and likely mediates trans-activation of a subset of IFNgamma -inducible early response genes.


MATERIALS AND METHODS

Cell Culture

As described previously (22), HK were isolated from neonatal foreskins at the Emory Skin Diseases Research Center. HK were cultured in KGM supplemented with bovine pituitary extract (Clonetics Corp., San Diego, CA). Cultures were maintained at 37 °C in humidified 5% CO2 and passaged at 60-70% confluence using subculture reagents from Clonetics. Experiments with HK were conducted with cells in passage 3.

Cytoplasmic and Nuclear Extract Preparation

Cytoplasmic and nuclear extracts were prepared as described previously (23) from cells that were either left untreated or treated with 250 units/ml recombinant human IFNgamma (R&D Systems, Minneapolis, MN). Cells were washed twice in ice-cold phosphate-buffered saline (Life Technologies, Inc.), then quickly washed in buffer A (10 mM Hepes, pH 7.4, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol (DTT), 50 µM phenylmethylsulfonyl fluoride (PMSF); all from Sigma). After centrifugation at 2,000 rpm (4 °C for 5 min), the cell pellet was resuspended in buffer A containing 0.1% Nonidet P-40 (U. S. Biochemical Corp.) and incubated on ice for 10 min. The nuclei were collected by centrifugation at 4,000 rpm for 2 min at 4 °C. The supernatant (cytosolic fraction) was removed and saved, and the pellet was resuspended in buffer B (20 mM Hepes, pH 7.4, 1.5 mM MgCl2, 420 mM NaCl, 1 mM DTT, 50 µM PMSF, 0.2 mM EDTA, 25% glycerol). The nuclear pellet was incubated on ice for 30 min, followed by centrifugation at 14,000 rpm for 15 min. The protein concentrations of the cytosolic and nuclear fractions were determined using UV absorbance at 280 nm as described (24). Proteins were used immediately in a binding reaction or aliquoted and stored at -70 °C.

Electrophoretic Mobility Shift Assay (EMSA)

The DNA binding reaction was performed for 30 min at room temperature in a volume of 20 µl, containing 5 µg of nuclear or cytoplasmic protein extract, 2.5 µg of bovine serum albumin (Life Technologies, Inc.), 2 µg of poly(dI-dC) (Sigma) 5 µl of 4 × binding buffer (1 × buffer: 12 mM Hepes, pH 7.8, 4 mM Tris, 60 mM KCl, 1 mM EDTA, 12% v/v glycerol, 1 mM DTT, 1 mM PMSF) with or without 10-100-fold molar excess of cold competitor DNA. Radiolabeled probe (1 × 105 cpm) was added for an additional incubation period of 20 min. In supershift EMSA, cytoplasmic or nuclear extracts were incubated with experimental or isotype control antibody, at supplier's recommended concentrations, prior to the addition of the 32P-labeled probe. Polyclonal antibodies to Stat1 and monoclonal antibodies to Stat1 through Stat6 and to p48 were obtained from Transduction Laboratories, Lexington, KY. Polyclonal antibodies to both the amino and carboxyl termini of Stat1 were kindly provided by J. E. Darnell, Rockefeller University, New York, NY. DNA binding reactions were separated on 4% native polyacrylamide gels. Gels were subsequently dried and autoradiography performed. The autoradiographs were scanned on a La Cie flat bed scanner (La Cie Ltd., Beaverton, OR) utilizing Adobe Photoshop software (Adobe Systems, Inc., Mountain View, CA). Subsequently the digitized image was labeled in Microsoft Power Point (Microsoft Corp., Redmond, WA) and printed on a high resolution laser printer. Each figure represents a computer-generated image of the autoradiograph, and each is typical of the autoradiograph in the context of relative band and background densities.

To test the requirement of tyrosine phosphorylation for DNA-protein complex formation, nuclear protein extracts were exposed to 10 units of protein-tyrosine phosphatase (PTPase) 1-B from Yersinia pestis (Upstate Biotechnology, Inc., Lake Placid, NY) in the presence or absence of the PTPase inhibitor sodium orthovanadate (1 mM). These reactions were carried out for 30 min at 30 °C in a 15-µl reaction containing 20 mM Tris-HCl, pH 7.4, 0.5 mM DTT, 0.1 mM EGTA. Treated extracts were then incubated with 32P-labeled pIgamma RE as probe and analyzed by EMSA.

All oligonucleotides used for probes or as cold competitors were synthesized at the Emory University Microchemical Facility. Double-stranded oligonucleotides were prepared by annealing complementary strands as described (25). The pIgamma RE oligonucleotide was synthesized to include the IFNgamma -responsive site (in bold letters) found in the ICAM-1 gene promoter (5'-<UNL>CGAAGCT</UNL>TTTCCGGGAAA<UNL>GGATCCC</UNL>-3'). The underlined sequences in the pIgamma RE oligonucleotide represent restriction sites that were used to create overhangs for labeling with 50 µCi of [alpha -32P]dCTP (DuPont NEN) by fill-in reaction using Klenow (Stratagene, La Jolla, CA) as described (25). Alternatively, a short primer, GGGATCCTTTCC, complementary to the 3' end of the above sequence was used to label pIgamma RE by a primer-extension fill-in reaction using Klenow and 50 µCi of [alpha -32P]dCTP (25). Unincorporated nucleotides were removed by column chromatography over G25 Sephadex columns (Boehringer Mannheim). The [alpha -32P]dCTP-labeled probe was used in analysis of the phosphatase-treated or untreated proteins in EMSA. In all other EMSAs 5' end-labeled probes were prepared with 100 µCi of [gamma -32P]ATP (Amersham Corp.) using T4 polynucleotide kinase (New England Biolabs, Beverly, MA) and were gel-purified as described (25).

Oligonucleotides corresponding to IFNREs (written in bold letters) of the genes indicated were used as radiolabeled probes or as cold competitors. For all oligonucleotides used, non-wild type restriction sites (underlined) were incorporated flanking the identified REs for cloning and labeling purposes. Oligonucleotides used were as follows: GAS of the GBP gene: <UNL>CGAAGCTT</UNL>AGTTTCATATTACTCTAAATC<UNL>GGATCCC</UNL> (26); ISRE of the GBP gene: <UNL>CGAAGCTT</UNL>CGAAGTACTTTCAGTTTCTATTA<UNL>GGATCCC</UNL> (9); ISRE of the 6-16 gene: <UNL>CGAAGCTT</UNL>CCCTTTTACTTTGA<UNL>GGATCCC</UNL> (27); GRR of the Fcgamma R1 gene: <UNL>CGAAGCT</UNL>TTTCTGGGAAA<UNL>GGATCCC</UNL> (28); pIRE of the ICSBP gene: <UNL>CGAAGCT</UNL>TTTCCGAGAAA<UNL>GGATCCC</UNL> (29); IFNRE of the IRF-1 gene: <UNL>CGAAGCT</UNL>TTTCGGGGAAA<UNL>GGATCCC</UNL> (30).

In experiments comparing complexes and mobility of pIgamma RE versus the GAS element, a short primer (GGGATCCGATTTAGA) complementary to the 3' end of the GAS sequence of the GBP gene, was used to label GAS by a primer-extension fill-in reaction using Klenow and 50 µCi of [alpha -32P]dCTP. Unincorporated nucleotides were removed by column chromatography over G25 Sephadex columns.

In addition, four oligonucleotides incorporating distinct mutations, insertions, or deletions in the wild type pIgamma RE sequence were also used as competitors or probes. These pIgamma RE mutants, with mutated nucleotides shown in italics, were: MUT 1, <UNL>CGAAGCT</UNL>TCACCGGGAAA<UNL>GGATCCC</UNL>; MUT 2, <UNL>CGAAGCT</UNL>TTTCCGGGAGA<UNL>GGATCCC</UNL>; MUT 3, <UNL>CGAAGCT</UNL>TTTCCATGCATGCATGGAAA<UNL>GGATCCC</UNL>; MUT 4, <UNL>CGAAGCT</UNL> TTTCC(X)GGAAA<UNL>GGATCCC</UNL>. Each mutant incorporates a distinct class of change; MUT 1 has a two-nucleotide mutation in the 5' side of the of pIgamma RE, MUT 2 has a one-nucleotide mutation in the 3' side of pIgamma RE, MUT 3 has a 10-bp insertion separating the 5' and 3' sides of the palindrome, and MUT 4 deletes the central G-C hinge in the palindrome. In addition, two different 30-bp sequences from regions of ICAM-1 located upstream from the pIgamma RE were used as irrelevant competitor DNA fragments.


RESULTS

IFNgamma Induces Activation of a Specific pIgamma RE-binding Factor (GRF) in a Time-dependent Manner

We examined by EMSA whether specific factors that can bind to pIgamma RE are activated in HK by IFNgamma treatment. As seen in Fig. 1A, stimulation of cells with IFNgamma led to the induction of a distinct DNA-binding protein complex (GRF), resulting in retarded mobility of the labeled pIgamma RE probe. However, no pIgamma RE binding activity was observed in nuclear extracts isolated from untreated HK. Binding of GRF to labeled pIgamma RE was not competed by excess unlabeled, irrelevant DNA as competitor, while excess unlabeled pIgamma RE competed for GRF binding, indicating the specificity of the retarded complex. In order to determine the kinetics of induction of GRF, HK were treated with IFNgamma for various periods of time before preparing both cytoplasmic and nuclear extracts for EMSA. As seen in Fig. 1B, the binding activity of cytoplasmic extracts from HK peaked after 30 min of IFNgamma treatment, decreased after 4 h, and declined significantly by 8 h. By 24 h, binding activity was not present. Stimulation of HK with IFNgamma led to the induction of GRF in both cytoplasmic as well as nuclear fractions within seconds (data not shown), consistent with the characteristic rapid activation of latent proteins by this cytokine (13).


Fig. 1. IFNgamma induces a specific pIgamma RE-binding GRF complex in HK cells in a time-dependent manner. A, EMSA using a 32P-end-labeled pIgamma RE probe incubated with 5 µg of total nuclear extracts from HK that were either untreated (lane 1) or IFNgamma -treated (lanes 2-5). A 10-fold molar excess of unlabeled competitor oligonucleotide was added as follows: distinct but irrelevant 30-bp double-stranded oligonucleotides (lanes 3 and 4) and pIgamma RE (lane 5). B, EMSA using a 32P-end-labeled pIgamma RE probe incubated with 5 µg of total cytoplasmic extracts from HK that were either untreated or treated with IFNgamma for specified times. Reactions with extracts from untreated cells are shown in lane 1, and reactions with extracts from cells treated with IFNgamma for varying times (in hours) are shown in lanes 2-6.
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pIgamma RE Is Necessary for GRF Binding Activity, and Mutated pIgamma RE Sequences neither Compete nor Bind to GRF

We next addressed whether targeted mutations, insertions, or deletions within the 11-bp pIgamma RE palindromic sequence had any effect upon GRF binding. As seen in Fig. 2A, mutations that disrupted either the 5' or the 3' end of the pIgamma RE palindrome failed to compete with wild type pIgamma RE for binding to GRF. Moreover, substitution with a 10-bp spacer (allowing for one full helical turn) between the palindromic halves or deletion of the G-C hinge of the palindrome abrogated the ability to compete with wild type pIgamma RE for binding to GRF. When used as radiolabeled probes, these mutated oligonucleotides also failed to display any retarded complexes upon incubation with lysates from IFNgamma -treated cells. Because the mutant pIgamma RE sequences failed to display any retarded complexes and failed to compete with pIgamma RE for GRF complex formation at 10-fold molar excess, we investigated whether these mutants displayed a concentration-dependent competition for GRF at higher molar ratios. Unlabeled mutant oligonucleotides ranging from 10-fold to 1000-fold molar excess were used in a competition EMSA. As seen in Fig. 2C, the mutants failed to display any competition with pIgamma RE for GRF complex formation even at a 1000-fold molar excess. Furthermore, unlabeled double-stranded oligonucleotide corresponding to the GAS sequence also failed to display competition for GRF complex formation when used at a 1000-fold molar excess. Finally, while the unlabeled double-stranded irrelevant oligonucleotide also failed to display competition for GRF at a 1000-fold excess, a competition EMSA utilizing unlabeled pIgamma RE displayed a detectable diminution of the labeled GRF complex at a unlabeled to labeled ratio of 0.01 to 1 (Fig. 2B), and a significant competition at 0.1 and 1 to 1 molar ratios (Fig. 2, B and C). These data demonstrate a high and specific binding affinity of pIgamma RE for GRF binding, a requirement for a specific sequence and spatial arrangement within this element, and a lack of competition by an oligonucleotide containing the classic GAS element previously demonstrated to bind homodimeric Stat1alpha .


Fig. 2. The pIgamma RE mutants do not compete for the pIgamma RE·GRF complex and do not form complexes with lysates from IFNgamma -treated HK. A, 32P-end-labeled pIgamma RE probe (lanes 1-7) or probes of pIgamma RE mutants MUT 1 (lanes 8 and 9), MUT 2 (lanes 10 and 11), MUT 3 (lanes 12 and 13) or MUT 4 (lanes 14 and 15) were incubated with nuclear extracts from HK that were either untreated (lanes 1, 8, 10, 12, 14) or IFNgamma -treated (lanes 2-7, 9, 11, 13, and 15). A 10-fold molar excess of unlabeled competitor DNA was added as follows: pIgamma RE (lane 3), MUT 1 (lane 4), MUT 2 (lane 5), MUT 3 (lane 6), and MUT 4 (lane 7). B, a concentration-dependent competition EMSA using a 32P-end-labeled pIgamma RE probe incubated with 5 µg of total nuclear extracts from HK that were either untreated (lane 1) or treated with IFNgamma (lanes 2-8). The molar ratio of unlabeled pIgamma RE added as competitor to labeled pIgamma RE is indicated in lanes 3-8. C, a concentration-dependent competition EMSA using a 32P-end-labeled pIgamma RE probe incubated with 5 µg of total nuclear extracts from HK that were either untreated (lane 1) or treated with IFNgamma (lanes 2-20). Unlabeled competitor DNA was added as follows: pIgamma RE (lanes 3 and 4), Fcgamma R1·GRR (lane 5), GBP·ISRE (lane 6), GBP·GAS (lanes 7 and 8), MUT 1 (lanes 9-11), MUT 2 (lanes 12-14), MUT 3 (lanes 15-17), and MUT 4 (lanes 18-20). The molar ratio of unlabeled competitor to labeled pIgamma RE is indicated in lanes 3-20.
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The changes in the pIgamma RE palindromic sequence that abrogated GRF binding and complex formation were consistent with additional transcriptional activation studies using reporter gene constructs. None of the described mutations were capable of driving reporter gene expression, while wild type pIgamma RE was sufficient to confer IFNgamma inducibility of heterologous reporter gene constructs (data not shown). These results indicate that GRF requires an intact pIgamma RE palindromic sequence to bind to pIgamma RE in vitro and pIgamma RE is necessary to function in vivo.

GRF Activation Is Dependent upon Tyrosine Phosphorylation

The importance of protein-tyrosine phosphorylation in the activation of trans-acting proteins involved in IFNgamma -induced transcription of other genes (19, 31) led us to investigate whether pIgamma RE binding activity induced upon IFNgamma treatment of HK was dependent upon tyrosine phosphorylation. Purified recombinant PTPase 1-B, isolated from Y. pestis, is a close homologue of the human tyrosine-specific phosphotase PTPase 1 and has been found to specifically catalyze the removal of phosphate from tyrosine residues (32). We assessed the effect of PTPase 1-B on the pIgamma RE·GRF complex formation. When the pIgamma RE probe was incubated with IFNgamma -treated HK cell lysates in the presence of PTPase 1-B, formation of the pIgamma RE·GRF complex was abrogated (Fig. 3). However, addition of the PTPase inhibitor sodium orthovanadate prior to incubation with the enzyme prevented disruption of complex formation, while the inhibitor itself had no effect upon the complex formation. These results indicate that tyrosine phosphorylation is needed for formation of the pIgamma RE·GRF complex, a property consistent with GRF containing a STAT-like protein (13).


Fig. 3. Activation of the pIgamma RE-binding complex by IFNgamma treatment of cells is dependent on tyrosine phosphorylation. EMSA using a Klenow filled-in 32P-labeled pIgamma RE probe incubated with 5 µg of cytoplasmic extracts from HK that were either untreated (lane 1) or IFNgamma -treated (lanes 2-5). Selected reactions (lanes 4 and 5) were preincubated with 10 units of PTPase 1-B prior to the addition of probe. 1 mM sodium orthovanadate (Na3VO4), was added to selected reactions (lanes 3 and 5) prior to the addition of both PTPase 1-B and probe. Locations of GRF and nonspecific bands (NS) are indicated.
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Anti-Stat1alpha Antibodies Interact with the GRF

Since IFNgamma activation of Stat1alpha is known to involve tyrosine phosphorylation of quiescent cytoplasmic proteins, we investigated the possibility of Stat1alpha involvement in GRF using anti-Stat1alpha antibodies in supershift EMSAs. As shown in Fig. 4, addition of polyclonal anti-Stat1alpha antibodies directed against either the amino-terminal or the carboxyl-terminal portions of Stat1alpha to IFNgamma -treated cell lysates supershifted the pIgamma RE·GRF complex, and these supershifted complexes were competed away by excess unlabeled pIgamma RE. Irrelevant antibodies had no effect upon complex formation or mobility (data not shown). Since polyclonal antibodies raised against both the amino- and the carboxyl-terminal portions of Stat1alpha supershift the pIgamma RE·GRF complex, and since Stat1beta lacks the 38 carboxyl-terminal amino acids that are included in Stat1alpha , it is extremely unlikely that the Stat1-like protein identified through these studies is Stat1beta . Monoclonal anti-Stat1alpha antibodies used in subsequent studies, while specific for Stat1alpha , were raised against peptide regions common to Stat1alpha and Stat1beta , and thus do not provide evidence to include or exclude Stat1beta as a possible component of the pIgamma RE·GRF complex. However, polyclonal antibody supershift data presented in Fig. 4 provide strong evidence that the GRF complex does not contain Stat1beta .


Fig. 4. Anti-Stat1alpha antibodies supershift the pIgamma RE·GRF complex. EMSA using a 32P-end-labeled pIgamma RE probe incubated with 5 µg of nuclear extracts from HK that were either untreated (lane 1) or IFNgamma -treated (lanes 2-7). Polyclonal anti-Stat1alpha antibodies directed against the amino-terminal (lanes 4 and 5) or the carboxyl-terminal (lanes 6 and 7) were added to extracts prior to the addition of probe. A 10-fold molar excess of unlabeled pIgamma RE was added (lanes 3, 5, and 7) as cold competitor. The pIgamma RE·GRF complex and the supershifted complex are indicated.
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Antibodies to Other STAT Proteins Do Not Supershift the pIgamma RE·GRF Complex

Since recent reports have shown that numerous STATs and non-STAT proteins interact with Stat1 in nuclear transcription complex formation (19), we investigated whether GRF contained any other known STAT proteins by supershift EMSA. As shown in Fig. 4, both polyclonal and monoclonal antibodies to Stat1alpha were able to supershift the pIgamma RE·GRF complex. However, antibodies to Stat2, Stat3, Stat4, Stat5, and Stat6 did not interact with this complex (Fig. 5). Antibodies to p48, a component of the ISGF3 complex, also did not react with the GRF (data not shown).


Fig. 5. The pIgamma RE·GRF complex is not recognized by antibodies to STAT proteins other than Stat1. EMSA using a 32P-end-labeled pIgamma RE probe incubated with 5 µg of nuclear extracts from HK that were either untreated (lane 1) or IFNgamma -treated (lanes 2-9). Anti-STAT antibodies were added as follows: polyclonal anti-Stat1alpha (amino-terminal, lane 3); monoclonal anti-Stat1alpha (amino-terminal, lane 4); and monoclonal antibodies to Stat2 (lane 5), Stat3 (lane 6), Stat4 (lane 7), Stat5 (lane 8), and Stat6 (lane 9). The locations of the probe·GRF complex (GRF), supershifted pIgamma RE·GRF-antibody complexes (A and B), and a nonspecific band (NS) appearing with untreated and IFNgamma -treated extracts are indicated.
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GRF Contains a Distinctly Different DNA-Protein Complex

In order to investigate further whether the anti-Stat1alpha antibody supershifted GRF complex contained a form of Stat1 that could be competed by GBP·GAS or GBP·ISRE, we performed competition supershift EMSA. As seen in Fig. 6, the supershifted GRF when bound to pIgamma RE was not competed by excess unlabeled double-stranded oligonucleotide corresponding to either the GAS or ISRE elements of the GBP gene. However, this complex was competed by excess of unlabeled pIgamma RE and was also competed by double-stranded DNA corresponding to the IFNRE of the IRF-1 gene, an IFNgamma RE that varies in sequence from pIgamma RE by only a single nucleotide (see Table I).


Fig. 6. The IFN response elements GAS and ISRE of the GBP gene and the IFNgamma RE of the IRF-1 gene display differential competition for GRF binding to pIgamma RE. EMSA using a 32P-end-labeled pIgamma RE probe incubated with nuclear extracts from HK either untreated (lane 1) or IFNgamma -treated (lanes 2-10). Anti-Stat1alpha monoclonal antibody (amino-terminal) was added in lanes 3-10, and a 10-fold molar excess of unlabeled competitor DNA was added as follows: pIgamma RE (lane 4), IRF-1 IFNRE (lane 6); GBP·GAS (lane 8), and GBP·ISRE (lane 10).
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Table I.

Comparison of the sequences and GRF binding capacities of wild type and mutated IFNgamma REs

Nucleotides in the IFNgamma REs that are different from those of ICAM-1 pIgamma RE are indicated by lowercase type. Mutated nucleotides in pIgamma RE sequences designated as MUT 1 through MUT 4 are indicated in italics. The insertion mutation of 10 bp (ATGCATGCAT) in ICAM-1 MUT 3 is indicated as (10 bases), and the deletion mutation of the central G in ICAM-1 MUT 4 is indicated as an (X). The binding capacity of each sequence to bind GRF is indicated as follows: sequences that can bind to GRF, +; sequences that do not bind to GRF, -.
Gene IFNRE Reference Sequence GRF binding capacity

Fcgamma RI GRR (28)    TTTCtGGGAAA +
ICSBP pIRE (29)    TTTCCGaGAAA +
IRF-1 IFNRE (30)    TTTCgGGGAAA +
ICAM-1 pIgamma RE (20, 22, Footnote 2)    TTTCCGGGAAA +
ICAM-1 MUT1 This study    TCACCGGGAAA  -
ICAM-1 MUT2 This study    TTTCCGGGAGA  -
ICAM-1 MUT3 This study    TTTCC(10 bases)GGAAA  -
ICAM-1 MUT4 This study    TTTCC(X)GGAAA  -
GBP GAS (26)    AGTTTCATATTACTCTAAATC  -
GBP ISRE (9)    CGAAGTACTTTCAGTTTCATATTA  -
6-16 ISRE (27)    CCCAAAATGAAACT  -
Ly6A/E GAS (41)    TTTCCtGtAAA  -

The pIgamma RE·GRF Complex Displays a Distinct Mobility in EMSA, and pIgamma RE Does Not Compete with the GAS·GAF Complex

Our data from the supershift EMSA using anti-Stat1alpha antibodies suggests that Stat1alpha , or a Stat1alpha -like protein, is part of the GRF that binds to the ICAM-1 pIgamma RE, but both the GAS and ISRE elements failed to compete with pIgamma RE for complex formation. Therefore, we investigated whether pIgamma RE could cross-compete with GAS. In addition, because Stat1alpha had previously been shown to bind to the GAS element of several genes as a homodimer referred to as GAF (8), we also investigated whether the complexes formed with pIgamma RE and GAS displayed any differences in EMSA. Using pIgamma RE or GAS as probes with IFNgamma -treated HK cell lysates, we observed striking differences in the mobility of complexes formed with pIgamma RE and GAS when run in the same gel (Fig. 7). GRF displayed a distinctly slower mobility compared to the GAF complex. Furthermore, unlabeled pIgamma RE at a 1000-fold molar excess failed to compete with GAS for the binding of GAF complex. However, a 1 to 1 molar ratio of unlabeled ISRE displayed significant competition with labeled GAS for binding GAF, while a 10-fold molar excess of unlabeled ISRE completely competed for GAF·GAS complex formation. More importantly, unlabeled GAS displayed a concentration-dependent competition for binding of GAF with a significant diminution of the labeled GAS·GAF complex when unlabeled GAS was added at only a 0.1 to 1 molar ratio. However, neither unlabeled GAS or ISRE, when added to labeled pIgamma RE reactions, displayed any ability to compete for GRF complex formation even when added at a 1000-fold molar excess (Fig. 7). Interestingly, excess unlabeled IFNRE of the IRF-1 gene displayed a similar concentration-dependent competition for GRF complex formation to that displayed by unlabeled pIgamma RE, suggesting similar binding affinities of GRF to these two IFNgamma -responsive elements, as was the case using the Fcgamma R1·GRR element as competitor for GRF binding in earlier experiments (Fig. 2C). These results indicate that GRF, which complexes with pIgamma RE of ICAM-1, IFNRE of IRF-1, and GRR of Fcgamma R1, is distinct from the classic GAF complex that binds to GAS.


Fig. 7. pIgamma RE·GRF and GAS·GAF are distinct complexes. 32P primer-extension labeled pIgamma RE or GAS probes were incubated with 5 µg of nuclear extracts from HK that were either untreated (lanes 1 and 13) or IFNgamma -treated (lanes 2-12 and 14-20). Complexes formed with labeled pIgamma RE (GRF) are shown in lanes 1-12, and complexes formed with labeled GAS (GAF) are shown in lanes 13-20. Unlabeled competitor DNA was added as follows: irrelevant (lanes 3 and 4), IRF-1 IFNRE (lanes 5-8), GBP·GAS (lanes 9, 10, and 18-20), GBP·ISRE (lanes 11, 16, and 17), pIgamma RE (lane 12 and 15). The molar ratio of the unlabeled competitor oligonucleotide to labeled probe is indicated in lanes 3-12 and 15-20.
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GRF Displays Similarity with the DNA-Protein Complex Formed with the IFNgamma RE of the Fcgamma R1 Gene

In addition to the ability of IFNRE of the IRF-1 gene to compete for GRF binding to pIgamma RE (Fig. 6 and 7), studies in our laboratory revealed that the IFNgamma response element pIRE of the ICSBP gene (29) also functioned well as a competitor for GRF binding (data not shown). We therefore investigated whether the complexes formed with pIgamma RE and those formed with a representative element of those genes that contain IFNgamma REs with sequences very similar to pIgamma RE (see Table I and below) displayed similar or different mobilities in EMSA. Using labeled oligonucleotides of equal size (25 bp) containing either pIgamma RE or the GRR of the Fcgamma R1 gene (28) as probes, we compared EMSA mobilities of the DNA-protein complexes formed when these probes were incubated with IFNgamma -treated HK cell lysates. As seen in Fig. 8, the complexes that formed with pIgamma RE and GRR displayed similar mobility. GRR also competed for GRF binding to pIgamma RE, as seen in Fig. 2C, and unlabeled pIgamma RE displayed a similar competition for GRF binding to GRR (data not shown). In addition, both complexes were supershifted in a similar manner with monoclonal antibodies to Stat1alpha .


Fig. 8. The pIgamma RE·GRF complex displays similar electrophoretic mobility and anti-Stat1 reactivity as the DNA-protein complex formed with the IFNgamma RE (GRR) of the Fcgamma R1 gene. 32P-End-labeled Fcgamma R1·GRR probe (lanes 1-3) or pIgamma RE probe (lanes 4-6) was incubated with nuclear extracts from HK that were either untreated (lanes 1 and 4) or IFNgamma -treated (lanes 2, 3, 5, and 6). Anti-Stat1alpha monoclonal antibody (amino-terminal) was added to reactions prior to addition of probe (lanes 3 and 6). The locations of pIgamma RE·GRF and Fcgamma R1·GRR-protein complexes (GRF) and anti-Stat1alpha supershifted complexes are indicated.
[View Larger Version of this Image (46K GIF file)]


Taken together, the data presented in these experiments indicate that pIgamma RE of the ICAM-1 gene, GRR of the Fcgamma R1 gene, IFNRE of the IRF-1 gene, and pIRE of the ICSBP gene form a subset of IFNgamma response elements that bind to a trans-activating complex, gamma  response factor, that contains a Stat1alpha -like protein. This GRF complex clearly appears to be distinct from the homodimeric Stat1alpha complex that forms GAF and binds to the GAS element of other IFNgamma -responsive genes, such as that characterized in the guanylate-binding protein gene. Comparison of the identified critical sequences of these GRF-binding and non-GRF-binding elements and their distinct differences, as well as the mutations used in the above studies, are shown in Table I. These data and comparisons indicate that a potential consensus sequence, TTTCNGNGAAA, is required for binding of GRF. They also indicate that elements such as GAS and ISRE, which do not contain the characteristic 11-bp palindrome sequence displayed by pIgamma RE, bind to Stat1alpha containing complexes but not to the complex typified by the gamma  response factor, which binds to pIgamma RE and similar elements identified in a subset of IFNgamma -responsive genes.


DISCUSSION

We have characterized a specific DNA-binding complex, which we have termed GRF, that binds to the minimal IFNgamma -responsive element of the ICAM-1 gene, pIgamma RE. Formation and binding of GRF is dependent on IFNgamma -induced activation of pre-existing proteins, as demonstrated by the rapid activation and binding of GRF and by the activation of this complex in the presence of cycloheximide.3 In addition, we have demonstrated that the pIgamma RE sequence is necessary for binding GRF and that mutant pIgamma RE sequences neither bind GRF nor compete with pIgamma RE for the pIgamma RE·GRF complex even when used at high molar ratios. Moreover, pIgamma RE displays a high and specific binding affinity for GRF.

IFNgamma signaling involves activation of Stat1 by phosphorylation of a tyrosine residue in order to assemble active transcription-stimulating complexes (33, 34). Treatment with PTPase 1-B, which specifically dephosphorylates tyrosine residues, has been shown to abrogate binding of these transcription complexes as shown by EMSA (34, 35). Our data indicate that IFNgamma -induced activation of GRF is tyrosine phosphorylation-dependent as well.

It has been shown that Stat1alpha is activated by a number of other agonists, in addition to IFNgamma and IFNalpha , based largely on reactivity of binding complexes with anti-Stat1alpha antibodies (19, 36, 37, 38). Our supershift data using antibodies to the known STAT proteins suggest that Stat1alpha , or a protein with antigenic similarities to Stat1alpha , is a component of GRF. In addition, studies using polyclonal antibodies to either the amino-terminal or the carboxyl-terminal portions of Stat1alpha appear to exclude Stat1beta as a possible component of the GRF complex. These data are in agreement with the recently reported GAF-like factor binding to the IFNgamma RE of ICAM-1 gene (39) and the recent finding of Stat1alpha involvement in the complex by immunoblotting using anti-Stat1 antibodies (40). Previous reports indicate that Stat1alpha is a common component of the IFN-activated trans-acting complexes GAF and ISGF3. Stat1alpha binds to GAS as the homodimer GAF, and the multimeric ISGF3 complex, which binds to ISRE, contains Stat1alpha in addition to Stat2 and p48 (19). However, in the present studies, neither a double-stranded oligonucleotide corresponding to GBP·GAS nor GBP·ISRE used as unlabeled competitors competed with pIgamma RE for the binding of GRF. Further, we have observed that double-stranded oligonucleotides corresponding to the IFNgamma REs of the ISRE from the 6-16 gene (27) and the GAS sequence (GCGGATCCTTTCCTGTAAAAGCTTGC) from the Ly6A/E gene (41) also fail to compete with pIgamma RE for GRF complex formation.4

Our studies demonstrate a lack of cross-competition between GAS and pIgamma RE for formation of their respective DNA-protein complexes. However, ISRE from the GBP gene, which binds Stat1alpha as part of the heterotrimer in the ISGF3 complex and does not compete with pIgamma RE for complex formation, clearly competes with GAS for the GAS·GAF complex. We have observed that both GAS and ISRE do not compete with pIgamma RE even at a 1000-fold excess in our experiments using HK cell lysates. Our results are in agreement with the report that shows GBP·GAS does not compete with the ICAM-1 IFNgamma RE in airway epithelial cells (20) and are in contrast to those obtained by others in MeL JuSo cells (42).

Consistent with our results, GRF that is supershifted by anti-Stat1alpha antibodies is not competed away by excess unlabeled GAS or ISRE from the GBP gene. On the other hand, excess unlabeled IFNRE from the IRF-1 gene displayed competition for the supershifted GRF. In fact, semi-palindromic IFNgamma REs from other IFNgamma -responsive genes, such as the IFNRE of the IRF-1, pIRE of the ICSBP gene and GRR of Fcgamma R1 gene, show somewhat closer sequence homology with that of pIgamma RE than do the sequences of various GAS and ISRE elements as shown in Table I. It is precisely these elements that share greater sequence homology with pIgamma RE that can function as competitors in EMSAs with pIgamma RE for complex formation in IFNgamma -induced HK cell lysates. Interestingly, these REs show differences in their sequences in nucleotides immediately flanking the G-C hinge of the pIgamma RE. The mutations that rendered pIgamma RE completely nonfunctional in vitro and in vivo in our study are either in the 5' or 3' end of the palindrome or the G-C hinge of the palindrome, and suggest binding specificity of GRF for these specific sequences. Comparison of the sequences of IFNgamma REs of the IRF-1, ICSBP, and Fcgamma R1 genes with the pIgamma RE sequence of ICAM-1 and mutations of pIgamma RE sequence reveals a potential consensus sequence, TTTCNGNGAAA, that is required for binding of GRF. Among the GAS and ISRE sequences of genes such as GBP, 6-16, and Ly6A/E, the GAS sequence of Ly6A/E displays closest homology to this GRF-binding consensus sequence. However, the sequence of Ly6A/E diverges from the GRF-binding consensus sequence at two sites, the central G-C hinge and in the 3' half of the palindrome. We have been unable to demonstrate any competition for GRF binding with the LY6A/E·GAS. Whether this inability to bind GRF results from divergence of the nucleotide in the hinge or the 3' half of the palindrome has not yet been determined.

Finally, the significantly slower mobility of the pIgamma RE·GRF complex compared to that of the GAS·GAF complex, using identical IFNgamma -treated HK cell lysates with GAS and pIgamma RE as separate probes in the same EMSA, cannot be accounted for by differences in the sizes of the radiolabeled oligonucleotides. In fact, the smaller pIgamma RE probe (25 bp) formed a more slowly migrating DNA-protein complex when compared to the GAS·GAF complex bound by the larger (36 bp) GAS probe. In contrast, the IFNgamma -activated DNA-protein complexes formed with either the Fcgamma R1·GRR or the ICAM-1·pIgamma RE clearly display similar mobility on EMSA, and both complexes supershift in a similar fashion with anti-Stat1alpha antibodies. The IFNRE of the IRF-1 gene and the pIRE of the ICSBP gene form a DNA-protein complex with IFNgamma -treated HK cell lysates, which also displays a mobility similar to that of the pIgamma RE·GRF complex in EMSA (data not shown).

Other investigators have recently shown that Fcgamma R1·GRR does not compete with GBP·GAS in competition EMSA (43). Further, Fcgamma R1·GRR was also shown to bind a Stat1alpha -like protein that interacted with an additional 43-kDa protein in response to IFNgamma -stimulation (35, 44). The semi-palindromic IFNgamma RE of the MIG gene, which displays significant sequence homology to ICAM-1·pIgamma RE, has been reported to bind an IFNgamma -activated trans-activating factor (gamma RF-1) that is composed of at least two proteins of 95 and 130 kDa (45). Furthermore, gamma RF-1 was shown to exhibit differences in electrophoretic mobility distinct from GAF and to contain one or more subunits antigenically related to Stat1alpha (45).

These data thus indicate that pIgamma RE represents a distinct subset of IFNgamma REs found in a number of early response genes that mediate trans-activation in response to IFNgamma signaling through a DNA-binding protein complex (GRF) that is distinctly different from the previously characterized IFNgamma -activated complex, GAF. While GRF certainly appears to contain a Stat1alpha -like protein, identification and characterization of all of the components of this distinct trans-activating complex, their relationship to other STAT and non-STAT proteins, and the specific biochemical pathways involved in their activation will further elucidate the molecular mechanisms by which IFNgamma initiates differential responses at sites of localized inflammation.


FOOTNOTES

*   This work was supported by United States Public Health Service (PHS) Grants AR 41206 and AR 42687 (to S. W. C.), PHS Supplemental Research Grant to AR 41206 (to S. M. N.), Dermatology Foundation fellowship grants (to L.-J. L. and N. S.), and an Emory University Research Committee research grant (to S. M. N.). 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.
Dagger    Present address: Yamanashi Medical University, Dept. of Dermatology, 1110 Tamaho-cho Nakakoma-Gun, Yamanashi-Ken 40938, Japan.
§   Present address: Dept. of Dermatology, 2 Rhoads Pavillion, University of Pennsylvania, Philadelphia, PA 19104.
   To whom correspondence and reprint requests should be addressed: The Emory Skin Diseases Research Core Center, Dept. of Dermatology, 5001 Woodruff Memorial Bldg., Emory University School of Medicine, Atlanta, GA 30322.
1    The abbreviations used are: ICAM-1, intercellular adhesion molecule-1; IFNgamma , interferon gamma ; pIgamma RE, palindromic interferon gamma  response element; EMSA, electrophoretic mobility shift assay; STAT, signal transducers and activators of transcription; PTPase 1-B, protein-tyrosine phosphatase 1-B; GAS, gamma -activated sequence; ISRE, interferon-stimulated response element; GBP, guanylate-binding protein; HK, human keratinocytes; GAF, gamma -activated factor; bp, base pair(s); ISGF, interferon-stimulated gene factor; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; RE, response element; IFNRE, IFN response element.
2    N. Shibagaki, and S. W. Caughman, unpublished results.
3    L.-J. Li, and S. W. Caughman, unpublished results.
4    K. L. Quinlan, and S. W. Caughman, unpublished results.

Acknowledgments

We thank Dr. J. E. Darnell for the kind gift of polyclonal anti-Stat1 antibodies and Dr. Josiah N. Wilcox for invaluable assistance in preparation of the figures for this manuscript. We gratefully acknowledge the advice, technical help, and critical review of this manuscript by Dr. Johannes Bauer, Georgetta Cannon, Dr. Jennifer Duff, Dr. Tri Nguyen, and Virginia Secor.


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