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The Journal of Immunology, 2001, 166: 1041-1048.
Copyright © 2001 by The American Association of Immunologists

High Level Class II trans-Activator Induction Does Not Occur with Transient Activation of the IFN-{gamma} Signaling Pathway1

Donna D. Eason* and George Blanck2,*,{dagger}

* Department of Biochemistry and Molecular Biology, College of Medicine, and {dagger} Immunology Program, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, FL 33612


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Gene activation in early development is highly dependent on precise concentrations of trans-acting factors for the activation of different genes at differing points in the embryo. Thus, not only is the presence or absence of a particular trans-activator or repressor relevant in determining gene activation, but also the concentration of the regulatory protein must be above or below a certain threshold for proper gene regulation. Signaling pathways in somatic cells are thought to represent cascades of on/off switches, mediated most commonly by phosphorylation. Here we demonstrate a quantitative mechanism for regulating the level of a component of the IFN-{gamma} signaling pathway that in effect represents the differential sensitivities of STAT1, IFN-regulatory factor-1, and class II trans-activator (CIITA) to IFN-{gamma}. Unlike developmental gene regulation, in which specificity of gene activation is a function of regulatory protein concentrations, specificity of gene activation in the IFN-{gamma} signaling pathway is regulated by the duration of the activation of the primary IFN-{gamma}-regulatory protein, STAT1. This result most likely explains previously reported data indicating that a minimum amount of IFN-{gamma} is required for MHC class II gene activation despite the fact that the level of the IFN-{gamma}-inducible factor directly required for MHC class II induction, CIITA, directly correlates with the level of MHC class II expression. The induction of a high level of CIITA is dependent on sustained IFN-{gamma} signaling. The possible implications of this result for tumorigenesis are discussed.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Gene expression during early development is dependent on threshold levels of transcription factor concentration, such that graded signals in embryogenesis are ultimately converted into discrete cell fates (1, 2). Cytokines and growth factors activate signaling pathways through receptor-associated triggering of tyrosine phosphorylation in the somatic cells of fully formed organisms that mediate continued cellular differentiation, cell proliferation, and cell survival. The downstream responses are thought to result from cascades of on/off switches where each step represents a saturated pattern of activation. Specificity of gene activation is thought to occur through interactions between components of different pathways resulting in integrated signaling networks (3).

IFN-{gamma} is a pleiotropic cytokine released from activated T cells and NK cells (4, 5), causing growth suppression, immune modulation, and induction of apoptosis via the JAK/STAT signaling pathway. IFN-{gamma} binding to its receptor leads to phosphorylation and activation of STAT1, which transits to the nucleus to activate gene transcription (6). In some cases, these genes encode transcription factors, such as IFN-regulatory factor-1 (IRF-1),3 that mediate the downstream effects of STAT1 activation (Fig. 1GoA). In most cell types, IFN-{gamma} induces the expression of MHC class II molecules, cell surface heterodimers that present Ags to CD4+ T cells (7, 8). This report examines the quantitative relationship between the levels of components of the IFN-{gamma} signaling pathway leading to MHC class II induction, including IFN-{gamma} itself, STAT1, IRF-1, and the class II trans-activator (CIITA) (Fig. 1GoA). Our study was prompted by two observations: 1) as noted below, numerous human tumor lines express relatively high levels of STAT1 and IRF-1 but do not express detectable levels of CIITA, where one would expect a proportional CIITA activation; and 2) a minimum, relatively high level of IFN-{gamma} treatment is required for MHC class II induction (9), but when CIITA is expressed under the control of an artificially inducible promoter, the level of CIITA directly correlates with the level of MHC class II (10). Results described below indicate that the sole point of regulation that represents a relative insensitivity to IFN-{gamma} and that leads to MHC class II expression occurs at the point of CIITA induction. The lack of sustained induction of CIITA associated with low levels of IFN-{gamma} correlates with a transient, although saturating, STAT1 response. This contrasts with the point in the pathway directly preceding CIITA induction, IRF-1 induction, which is directly dependent on STAT1 function at the IRF-1 promoter and which increases proportionally to STAT1 activation. The dependence on the duration of STAT1 activation for sustained, high level CIITA promoter activation also contrasts with the HLA class II promoter activation, which is directly proportional to CIITA expression (10).



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  		FIGURE 1. The IFN-{gamma} signaling pathway and RT-PCR and Southern blot analysis for CIITA. A, The basic IFN-{gamma} signaling pathway leading to MHC class II expression. Latent, cytoplasmic STAT1 molecules are activated after IFN-{gamma} binding of the receptor. Activated STAT1 translocates to the nucleus and induces expression of IRF-1. STAT1 and IRF-1 comediate expression of CIITA (23 ), the MHC class II trans-activator. B–D, RT-PCR was performed with total cytoplasmic RNA isolated from cells treated with 400 U/ml IFN-{gamma} for 48 h or left untreated. The CIITA-inducible breast carcinoma cell line MDA468-S4-MTRb-1 (referred to as Rb1) (13 ) and the CIITA-constitutive expressing Raji B cell line served as positive controls. The RNA was reverse transcribed with random primers and amplified by 40 cycles of PCR. The PCR products were subjected to Southern blot analysis and detected with 32P-labeled CIITA-specific probe (see Materials and Methods). B, PCR using CIITA-specific primer set A, which amplifies the CIITA cDNA 5'-region. C, PCR using CIITA-specific primer sets B and C, which amplify the CIITA 3'- and central regions, respectively. D, Semiquantitative PCR using {gamma}-actin primers and 15 cycles of amplification, which represents the linear portion of the amplification curve. Arrows, positions of the PCR products. The cell lines and IFN-{gamma} treatments are as indicated.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and culture

The human non-small cell lung carcinoma (NSCLC) cell line H1734 (provided by Dr. Frederick J. Kaye) (11, 12), the bladder carcinoma cell line 5637 (ATCC HTB9), and the retinoblastoma cell lines Weri (Weri-Rb1) and Y79 (13) were maintained in RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 10% FCS (Fisher, Pittsburgh, PA), glutamine, antibiotics, and pyruvate. The Rb-transformed breast carcinoma cell line MDA-468-S4-MTRB-1 (referred to as Rb1) (13) was grown in DMEM/F12 supplemented with 10% calf serum (Fisher), 10-8 M mouse epidermal growth factor (Boehringer Mannheim), glutamine, and antibiotics. The Raji B cell line was maintained in RPMI 1640 containing 10% calf serum, pyruvate, and antibiotics. All cells were grown at 37°C in 7% CO2.

Reagents

IFN-{gamma} was obtained from Genzyme (Cambridge, MA). Abs against IRF-1 (C-20), and STAT1 (C-136) were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). The anti-phosphotyrosine STAT1 Ab was obtained from Upstate Biotechnology (Lake Placid, NY). HRP-conjugated secondary Abs were purchased from Amersham (Arlington Heights, IL).

Reverse transcription and RT-PCR

For RT-PCR analysis, total cytoplasmic RNA was prepared by the Nonidet P-40 lysis method (14) from cells that were left untreated or were treated for 48 h with 400 U/ml IFN-{gamma} (Genzyme). RNA (5 µg from each sample set) was primed with random hexamers (Life Technologies) and reverse transcribed with Superscript reverse transcriptase according to the manufacturer’s instructions (Life Technologies). PCR was performed in a 50-µl reaction mixture (10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.001% (w/v) gelatin, 5% DMSO, 0.2 mM dNTPs, 5 µl of the reverse transcribed product, and 10 pmol each CIITA-specific primer or {gamma}-actin primers. For the PCR step, the samples were overlaid with mineral oil, heated to 95°C for 2 min, and cooled to 85°C; and 1.25 U of Taq polymerase (Fisher) were added. For CIITA, PCR cycles were performed 40 times at 95°C for 1 min, 55°C for 1 min, and 72°C for 2 min with a 6-min final extension at 72°C. For semiquantitative PCR, 15 cycles of PCR were performed with {gamma}-actin primers.

Primers

The primers used for amplification of human CIITA cDNA were described in Ref. 15 . The CIITA-specific primers consisted of set A (the sense primer, nt 104–131; the antisense primer nt 812–791), set B (the sense primer nt 2288–2309; the antisense primer 3566–3539), and set C (the sense primer nt 671–692; the antisense primer nt 1536–1515). The sequences of the human {gamma}-actin primers were: sense primer (5'-CCCGGTGCTTCTGACCGAGGCC-3'); and antisense primer (5'-CAGACTGAGTA-CTTGCGCTC-3').

Southern blot analysis of CIITA

Twenty-five microliters of each PCR product were electrophoresed on 1% agarose gels and capillary transferred to nylon membranes (Hybond; Amersham Pharmacia, Piscataway, NJ). The membrane was hybridized with 200 ng random [32P]cDNA probe representing nucleotides 174 to 3048 of the cDNA and including one 479-bp intron of CIITA (15) in a 50% formamide solution containing 6x SSC, 32 mM Na2HPO4, 13 mM NaH2PO4, 0.2% SDS, 5x Denhardt’s solution, and 5 µg/ml denatured salmon sperm DNA at 42°C overnight. The blots were washed sequentially in solutions of 2x SSC, 0.1% SDS for 30 min at 55°C, 0.5 x SSC, 0.1% SDS for 30 min at 65°C, and 0.2x SSC/0.5% SDS at 65°C for 30 min and exposed to x-ray film at -70°C.

Preparation of nuclear extracts

Cells were plated on either 100-mm or 150-mm plates at densities sufficient to reach ~75% confluency within 2 days. The cells were then left untreated or were treated for the designated time periods with 400 U/ml IFN-{gamma} (Genzyme), and nuclear and cytoplasmic extracts were prepared as described in Ref. 16 . Briefly, adherent cells were rinsed on the plate twice with 5 ml ice-cold PBS and once with 5 ml cold PBS containing 1 mM Na3VO4 and 5 mM NaF. The cells were then rinsed with 2 ml hypotonic buffer (20 mm HEPES (pH 7.9), 1 mM EDTA, 1 mM EGTA, 20 mM NaF, 1 mM Na3VO4, 1 mM Na4P2O7, 1 mM DTT, 1x Complete Protease Inhibitor Cocktail (Boehringer Mannheim, Indianapolis, IN)) and scraped into 300 µl (100-mm plate) or 500 µl (150-mm plate) hypotonic buffer containing 0.2% Nonidet P-40. Suspension cells were collected with the media, pelleted at 3000 rpm for 5 min at 4°C, washed as above by resuspending and repelleting the cells, and then resuspended in the appropriate amount of hypotonic buffer. The cells were incubated on ice for 10 min, and the nuclei were pelleted at 15,000 rpm for 20 s. The supernatant, representing the cytoplasmic fraction, was removed to a fresh tube, aliquoted, and stored at -70°C. The nuclei were resuspended in either 50 µl (100-mm plate) or 100 µl (150-mm plate) high salt buffer (420 mM NaCl, 20 mM HEPES (pH 7.9), 1 mM EDTA, 1 mM EGTA, 20% glycerol, 20 mM NaF, 1 mM Na3VO4, 1 mM Na4P2O7, 1 mM DTT, and 1x Complete Protease Inhibitor Cocktail (Boehringer Mannheim)) and incubated at 4°C for 45 min on a rotating wheel. The cellular debris was pelleted by centrifugation at 12,000 rpm for 20 min at 4°C, and the supernatant was removed to a fresh tube, aliquoted, and stored at -70°C. Protein concentrations were determined using the bicinchonic acid protein assay reagent according to the manufacturer’s instructions (Pierce, Rockford, IL).

EMSAs

The hSIE probe representing the high affinity Stat binding site (SIEm67) described in Ref. 17 was synthesized (Integrated DNA Technologies, Coralville, IA), annealed by incubating equimolar ratios of complementary oligonucleotides, and labeled by Klenow fill-in as in Ref. 18 . EMSAs were performed as described in Ref. 16 with 5 µg nuclear lysate incubated with 0.5 ng of the 32P-labeled probes for 30 min at 30°C in a 20-µl reaction containing 10 mM HEPES (pH 7.9), 50 mM KCl, 1 mM EDTA, 5% glycerol, 0.5 mM DTT, 1 µg poly(dI-dC) (Sigma, St. Louis, MO), and 5 µg BSA (Boehringer Mannheim). The reactions were separated by electrophoresis on either 5 or 8% native polyacrylamide gels (39:1 polyacryla-mide:bisacrylamide) in 0.25x TBE running buffer (22.25 mM Tris, 22.24 mM borate, 0.5 mM EDTA) at 180 V for 2–3 h, fixed for 10 min in 10% methanol, 10% acetic acid, dried on a gel slab dryer, and exposed to x-ray film at -70°C.

Western blot

Nuclear and cytoplasmic extracts were separated by SDS-PAGE on 8 or 10% polyacrylamide gels and transferred to Immobilon P filters (Millipore, Bedford, MA) by semidry transfer according to the manufacturer’s instructions (Millipore). The filters were blocked for at least 1 h at room temperature or overnight at 4°C in 20 mM Tris base (pH 7.6), 137 mM NaCl (TBS) containing 5% nonfat dry milk (w/v) and 0.5% Tween 20. The membrane was then probed for 4 h at room temperature or overnight at 4°C with the appropriate Ab, rinsed three times for 5 min each with TBS containing 0.5% Tween 20, and incubated for 1 h at room temperature with the appropriate secondary Ab conjugated to HRP. The membranes were washed as above, and the respective proteins were detected by ECL (Amersham).

Transient transfections

Cells (4.4 x 105) plated on 35-mm plates were transfected the following day with the respective promoter luciferase constructs using Lipofectamine and Lipofectamine Plus reagent or Lipofectace according to the manufacturer’s instructions (Life Technologies). The cells were incubated for 3 h at 37°C, then supplemented with complete medium, and incubated further for 1 h. The transfected cells were then treated with the specified amounts IFN-{gamma}, incubated for either 6 or 24 h at 37°C, and lysed with passive lysis buffer according to the manufacturer’s instructions (Promega, Madison, WI). Luciferase assays were performed using luciferase assay buffer (Promega) and measured with a Turner Designs Luminometer (TD-20/20, Turner, Palo Alto, CA). 5637 cells were transiently transfected with 730 ng of the HLA DRA-pGL3 luciferase construct (DRA (19)) or the CIITA type IV-pGL3 luciferase construct (CIITA (18)) and treated with either 50 or 400 U/ml IFN-{gamma} or left untreated. Luciferase activity was determined after 24 h IFN-{gamma} treatment (DRA) or after 6 and 24 h IFN-{gamma} treatment (CIITA). H1734 cells were transiently transfected as described in Ref. 20 with either 365 ng (experiments 1 and 3) or 730 ng (experiment 2) of the CIITA type IV-promoter luciferase construct and either left untreated or treated with 400 U/ml IFN-{gamma}. Luciferase activity was determined 24 h post-IFN-{gamma} treatment.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Lack of detectable CIITA activation in immortalized cell lines with inducible STAT1 and IRF-1 responses to IFN-{gamma}

We previously identified a number of MHC class II noninducible tumor cell lines that specifically lack IFN-{gamma} inducibility of CIITA (21). To determine whether a low level of CIITA transcripts was detectable in these cells, RT-PCR was performed with total cytoplasmic RNA from cells treated for 48 h with 400 U/ml IFN-{gamma} using three sets of CIITA specific primers spanning different regions of the CIITA mRNA. Southern analysis using a CIITA-specific cDNA probe revealed CIITA products in the IFN-{gamma} MHC class II-inducible breast carcinoma cell line MDA468-S4-Rb1 (Rb1) and the constitutive CIITA-expressing B cell line Raji, using CIITA primer set A (Fig. 1GoB), set B (Fig. 1GoC), and set C (Fig. 1GoC). However, no CIITA mRNA was detected with any of the CIITA primer sets in the IFN-{gamma} treated NSCLC cell line, H1734 (Fig. 1Go, B and C) or the retinoblastoma cell lines, WERI-Rb1 and Y79 (Fig. 1Go, B and C).

The CIITA type IV promoter contains a gamma activation sequence (GAS) and an interferon-regulatory factor element (IRF-E) that are bound by STAT1 and IRF-1, respectively, and both factors are required for the IFN-{gamma} induction of CIITA (22, 23, 24, 25). IRF-1 and STAT1 are the only known proteins activated by IFN-{gamma} that are associated with CIITA promoter activation. IFN-{gamma}-induced IRF-1 was clearly detectable in the CIITA-noninducible cell lines H1734, WERI-Rb1, and Y79, although these levels were reduced compared with the positive control, the 5637 cell line, which expresses a high level of CIITA after treatment with IFN-{gamma} (26) (Fig. 2GoA). The levels of IRF-1 mRNA transcripts correlated exactly with the protein levels observed in the Western analysis (Fig. 2GoA, inset). These results suggest that the low levels of IRF-1 protein observed in the Western analysis of CIITA-noninducible cells are related to the lack of IFN-{gamma} induction of CIITA gene expression but do not explain why CIITA expression is not proportional to the IRF-1 expression levels.



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  		FIGURE 2. Western blot and EMSA analyses of IFN-{gamma}-induced IRF-1 protein and STAT1 DNA-binding activity. A, Nuclear extracts from cells treated with 400 U/ml IFN-{gamma} for 2 h or left untreated were assayed for IRF-1 by Western analysis. The CIITA-inducible bladder carcinoma cell line 5637 (HTB9) served as a positive control. Protein samples were separated by 10% SDS-PAGE, transferred to polyvinylidene difluoride, and incubated with anti-IRF-1 Ab (diluted 1:1000; Santa Cruz Biotechnologies). The IRF-1 proteins detected by ECL are indicated by the arrow. The bar graph represents the percent of IFN-{gamma} induced IRF-1 mRNA () determined by phosphorimaging analysis of IRF-1 specific RPAs (27 ) and IRF-1 protein levels ({blacksquare}) in the four cells lines relative to the positive control 5637 cells (100%). B, EMSAs were performed with nuclear extracts from cells treated with 400 U/ml IFN-{gamma} for 15 min or 1 h or left untreated using the hSIE probe (17 ). Five micrograms of nuclear extract from each condition prepared as in Ref. 16 were incubated with the hSIE probe and subjected to EMSAs (18 ). The IFN-{gamma}-induced STAT1-DNA complex is indicated by the arrow. The cell lines and time of IFN-{gamma} treatments are as indicated. C, The nuclear extracts described in B were assayed for phosphotyrosine STAT1 protein by Western analysis. Protein samples were separated by 8% SDS-PAGE, transferred to polyvinylidene difluoride, incubated with anti-phosphotyrosine STAT1 Abs (Upstate Biotechnologies), and detected by ECL. The tyrosine-phosphorylated STAT1 proteins are indicated by the arrow. The cell lines and time of IFN-{gamma} treatments are as indicated. D, Cytoplasmic extracts from untreated cells were compared by Western blot as described above. The membrane was incubated with anti-STAT1 Ab (Santa Cruz Biotechnology), and the STAT1 proteins were detected by ECL. Top, Cell lines. The band representing STAT1 is indicated by the arrow.

 
To assay for IFN-{gamma}-induced phosphorylation of STAT1, which is required for the induction of IRF-1 gene transcription as well as CIITA transcription (22, 28), EMSAs were performed using nuclear extracts from cells treated with IFN-{gamma} for 15 min or 1 h. IFN-{gamma}-induced STAT1 DNA binding was observed in all of the extracts. The highest levels of activated STAT1 were observed in the CIITA-inducible positive control cell line, 5637 (Fig. 2GoB). The NSCLC cell line H1734 exhibited the lowest level of STAT1 DNA binding at both time points tested (Fig. 2GoB). The retinoblastoma cell lines WERI-Rb1 and Y79 had considerable STAT1 DNA binding activity after 15 min of IFN-{gamma} treatment, but DNA binding was diminished in the extracts from cells treated for 1 h with IFN-{gamma}, compared with the respective 5637 sample (Fig. 2GoB). Western analysis of cytoplasmic extracts indicated that the total STAT1 levels were identical in all of the tumor lines (Fig. 2GoD) and that the levels of phosphotyrosine STAT1 in nuclear extracts correlated with the DNA binding activity observed in the EMSA (Fig. 2GoC). Thus, STAT1 activation was clearly detectable in the CIITA noninducible lines with levels sufficient for activation of IRF-1 gene expression. However, these levels were not sufficient for the subsequent expression of CIITA as determined by the relatively sensitive RT-PCR/Southern blot assay (Fig. 1Go). The activation of STAT1 and the expression of IRF-1 are consistent with a previous report of detectable, albeit reduced, induction of the human guanylate-binding protein (hGBP) gene in these tumor lines (21). Like the CIITA promoter, the hGBP promoter requires binding of both STAT1 and IRF-1 for activation (29).

Low levels of IFN-{gamma} lead to a saturated STAT1 and IRF-1 response but do not lead to a sustained, high level CIITA response

We considered the possibility of mimicking the effect of partial activation of the IFN-{gamma} signaling pathway, apparent in the noninducible cell lines, by treating the CIITA-inducible cells with reduced amounts of IFN-{gamma}. 5637 cells were treated with increasing amounts of IFN-{gamma} and assayed for STAT1 DNA binding activity, IRF-1 protein levels, and CIITA mRNA levels at various time points. STAT1 DNA binding activity was induced at similar levels in cells treated for 15 min with 50, 100, 200, and 400 U/ml (Fig. 3Go, A and B). An increase in STAT1 DNA binding was observed that correlated with the increasing amounts of IFN-{gamma} in the cells treated for 1 h. After 2 h, STAT1 activation reached a plateau level that was similar for all concentrations of IFN-{gamma} (Fig. 3Go, A and B). Western analyses of these extracts revealed increasing IRF-1 protein levels with increasing amounts of IFN-{gamma} after 1 h (Fig. 3GoC), which increased further to similar levels for all of the extracts after 2 h of IFN-{gamma} treatment (Fig. 3GoC).



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  		FIGURE 3. STAT1 DNA-binding activity, IRF-1 protein, and CIITA mRNA induced by increasing concentrations of IFN-{gamma}. A, STAT1 DNA-binding activity was compared in nuclear extracts of 5637 cells treated for 15 min, 1 h, or 2 h with 50, 100, 200, or 400 U/ml IFN-{gamma}. Five micrograms of nuclear extract from each condition prepared as in Fig. 2GoB were incubated with the hSIE probe and subjected to EMSAs. Arrow, STAT1-bound probe. The lower band represents nonspecific binding (ns). Lane 1, free probe (P). B, The STAT1 DNA binding activity shown in A was quantified by phosphorimager scanning. The relative levels of STAT1 binding represent arbitrary units. C, IRF-1 protein levels induced by increasing concentrations of IFN-{gamma} in 10 µg nuclear extracts from 5637 cells treated for 1 or 2 h with concentrations of IFN-{gamma} as in A were separated by SDS-PAGE, transferred to polyvinylidene difluoride filters, and probed with an anti-IRF-1 Ab (Santa Cruz, as in Fig. 2GoA. D, RT-PCR and Southern analysis for CIITA mRNA induced by increasing concentrations IFN-{gamma} was determined using total cytoplasmic RNA prepared from 5637 cells treated with IFN-{gamma} as in A for 6 or 24 h or left untreated. Five micrograms of the total RNA were reverse transcribed (RT) using random primers; 20% of the RT reaction was amplified by PCR using CIITA-specific primers as in Fig. 1GoB, separated by electrophoresis, transferred to nylon filters, and probed with a 32P-labeled CIITA specific probe. The PCR and Southern analysis was repeated for the 24-h IFN-{gamma} treated extracts (experiments 1 and 2). E, Activation of HLA-DRA-promoter luciferase activity and CIITA type IV promoter activity with different IFN-{gamma} treatments (see Materials and Methods). 5637 cells were transfected with 730 ng of the HLA-DRA-pGL3 luciferase construct (DRA) or the CIITA type IV-pGL3 luciferase construct (CIITA) and treated with either 50 or 400 U/ml IFN-{gamma} or left untreated. Luciferase activity was determined after 24 h IFN-{gamma} treatment (DRA) or after 6 and 24 h IFN-{gamma} treatment (CIITA). Results are expressed as fold luciferase activity compared with the untreated sample from each condition. The results of the HLA-DRA-pGL3 experiment represent the mean value from six independent transfections. The results of the CIITA type IV-pGL3 are the mean value of three independent transfections.

 
To determine whether these IFN-{gamma} treatments were sufficient to activate CIITA gene expression, RT-PCR and Southern blot analysis was performed to assay for CIITA transcripts in cells treated for 6 or 24 h with increasing amounts of IFN-{gamma}. Low level CIITA transcripts were observed for all amounts of IFN-{gamma} in cells treated for 6 h with IFN-{gamma} (Fig. 3GoD) and are discussed below. However, after 24 h, no CIITA transcripts were detected in the RNA from cells treated with 50 U/ml IFN-{gamma} (Fig. 3GoD). Above the 50-U/ml concentration of IFN-{gamma}, it was possible to detect the CIITA mRNA (Fig. 3GoD). Thus, despite the nearly equivalent levels of STAT1 activation and IRF-1 synthesis induced by 50 and 400 U/ml after 2 h IFN-{gamma} treatment, only the 400-U/ml treatment led to a sizable and sustained induction of CIITA gene expression.

Sustained STAT1 activation is required for a sustained, high level CIITA response to IFN-{gamma}

To understand the basis for the lack of even low levels of CIITA expression at the 24-h time point, despite apparently saturating levels of activated STAT1 and IRF-1 produced by the lower doses of IFN-{gamma}, we considered the following: 1) occupancy of the CIITA promoter GAS element is observed relatively late after IFN-{gamma} treatment (24), although it is not known when the CIITA GAS element first becomes occupied; 2) IFN-{gamma} treatment of the WERI-Rb1 and Y79 tumor lines leads to high STAT1 activation levels at the early time point which decrease rapidly compared with the CIITA inducible positive control (Fig. 2GoB). These observations suggest the possibility that high level CIITA induction requires a sustained activation of STAT1. To test this possibility, IFN-{gamma} signaling was compared in cells treated with 50 and 400 U/ml at multiple time points. In addition, we transiently activated the pathway with a 15-min treatment of 400 U/ml IFN-{gamma} followed by withdrawal of the IFN-{gamma}. STAT1 activation was similar in extracts from cells treated for 2 h with 50 and 400 U/ml, as above, but decreased in the 6- and 24-h extracts representing 50 U/ml compared with those representing 400 U/ml IFN-{gamma} (Fig. 4GoA). STAT1 DNA binding activity was nearly identical in the extracts from cells treated with 400 U/ml IFN-{gamma} continuously for 2 h and the extracts from cells pulsed for 15 min (Fig. 4GoB). After 6 h, the STAT1 DNA-binding activity increased further in the cells continuously exposed to IFN-{gamma}, but not in the cells pulsed with a 15-min 400-U/ml IFN-{gamma} treatment (Fig. 4GoB). Sustained STAT1 DNA-binding activity was observed after 24 h in extracts from cells continuously exposed to 400 U/ml IFN-{gamma} (Fig. 4GoB), but not in extracts from cells receiving the short term IFN-{gamma} treatment. After 2 h IFN-{gamma} treatment, the IRF-1 levels appeared equivalent in all of the nuclear extracts (Fig. 4GoC). At 6 and 24 h, there was less IRF-1 in the cells treated with 50 U/ml and in the cells treated with 400 U/ml for 15 min, compared with the cells treated continuously with 400 U/ml (Fig. 4GoC). RT-PCR followed by Southern analysis detected CIITA transcripts in the 5637 cells treated continuously with 400 U/ml IFN-{gamma} for 24 h, but no transcripts were observed in cells treated with 50 U/ml or pulsed for 15 min with 400 U/ml IFN-{gamma} (Figs. 3GoD and 4D). Thus, a transient, saturating STAT1 response is not sufficient for high level CIITA activation. Sustained CIITA expression at least indirectly requires that the STAT1 activation be a sustained response.



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  		FIGURE 4. STAT1 DNA-binding activity, IRF-1 protein levels, and CIITA mRNA induced in cells treated for 2, 6, and 24 h with 50 and 400 U/ml IFN-{gamma}. A, The duration of STAT1 DNA binding activity at 2, 6, and 24 h by 50 or 400 U/ml IFN-{gamma} was compared in nuclear extracts of 5637 cells treated for 2, 6, and 24 h with 50 or 400 U/ml of IFN-{gamma} or left untreated. Five micrograms of nuclear extract from each condition were incubated with the hSIE probe and subjected to EMSAs as in Fig. 3GoA. Arrow, STAT1-bound probe. B, Comparison of STAT1 DNA-binding activity in cells treated for 2, 6, and 24 h with 50 or 400 U/ml or pulsed for 15 min with 400 U/ml IFN-{gamma}, then washed and incubated for the remaining total time of 2, 6, and 24 h. Nuclear extracts from each condition were analyzed as in A. C, The IRF-1 levels induced by IFN-{gamma} for 2, 6, and 24 h were determined in 20 µg nuclear extracts from 5637 cells treated for 2, 6, and 24 h with IFN-{gamma} as in A and subjected to Western analysis as in Fig. 3CGo. D, RT-PCR and Southern analysis for CIITA mRNA induced by continuous exposure to 50 U/ml, 400 U/ml, or a 15-min pulse with 400 U/ml IFN-{gamma}. Total RNA was prepared from 5637 cells treated with IFN-{gamma} as in A for 24 h or left untreated and analyzed by RT-PCR as in Fig. 3GoD.

 
As mentioned above, CIITA transcripts were detected in the cells treated with 50 U/ml IFN-{gamma} at 6 h but not after 24 h, indicating that the 6-h induction of STAT1 and IRF-1 was sufficient for initiation of CIITA transcription (Fig. 3GoD, lane 3). To verify the transient expression of CIITA under these conditions, 5637 cells were transiently transfected with a luciferase reporter gene driven by the HLA-DRA promoter, which requires CIITA for induction, then treated with either 50 U/ml or 400 U/ml IFN-{gamma} for 24 h, and assayed for luciferase activity (Fig. 3GoE). Luciferase activity was induced in the 50 U/ml IFN-{gamma}-treated samples, verifying the transient, low level induction of CIITA indicated by the RT-PCR assay, and reached ~20% of the levels induced by 400 U/ml IFN-{gamma} treatment (Fig. 3GoE, left). Next, 5637 cells were transiently transfected with the CIITA type IV promoter luciferase construct, then treated with either 50 or 400 U/ml IFN-{gamma} for 6 h or 24 h, and assayed for luciferase activity. The CIITA type IV promoter was activated at similar levels in both of the 6-h extracts (Fig. 3GoE, right). Luciferase activity was reduced in the extracts from cells treated with 50 U/ml IFN-{gamma} for 20 h, suggesting that the initial promoter activation was not sustained in the cells receiving 50 U/ml IFN-{gamma} (Fig. 3GoE, right). These results indicate that the transient, low level CIITA gene expression results, at least in part, from short term activation of the CIITA promoter by the IFN-{gamma}-induced transcription factors.

Despite the fact that transient activation of the upstream portion of the IFN-{gamma} signaling pathway (STAT1 phosphorylation and IRF-1 synthesis) does not result in high levels of sustained CIITA induction, the previous experiment indicated that partial activation of the pathway is sufficient to activate a promoter luciferase construct. Thus, the transfection of the CIITA promoter luciferase construct represents an assay for, or a record of, transient activation of the CIITA promoter where the partial activation of the signaling pathway may preclude a precise indication of when the transient activation of CIITA will occur or to what extent. Thus, we transfected the CIITA promoter luciferase construct into the CIITA-noninducible H1734 tumor line previously assayed by RT-PCR for CIITA induction at the 48-h time point (Fig. 1Go). This experiment revealed that the CIITA promoter is activated and indicates that the partial activation of the pathway observed in the tumor lines is sufficient for a transient activation of CIITA transcription at some point following IFN-{gamma} treatment (Fig. 5Go).



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  		FIGURE 5. IFN-{gamma} activation of CIITA type IV-promoter luciferase activity in H1734. H1734 cells were transiently transfected as described in Ref. 20 with either 365 ng (experiments 1 and 3) or 730 ng (experiment 2) of the CIITA type IV-promoter luciferase construct, and either left untreated () or treated with 400 U/ml IFN-{gamma} ({square}). Luciferase activity was determined 24 h post-IFN-{gamma} treatment. The results are expressed as fold luciferase activity compared with the untreated samples and are shown for each experiment. The CIITA promoter results represent three independent experiments. In experiment 3, the values represent the mean value of triplicate plates (±SEM).

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Sustained activation of a somatic cell signaling pathway

The results of this study indicate that the duration of IFN-{gamma} signaling plays a role in regulating specific gene activation. This work reveals a second mechanism, in addition to cross-talk between signaling pathways, for the regulation of signal transduction. The knowledge that specific genes can be regulated by the duration of the activation of a signaling pathway provides an opportunity for understanding certain phenotypes, previously noted as dependent on the duration of the activation of a signaling pathway, in terms of the transcriptional activation of genes. For example, sustained activation of the MAPK pathway is required for cell proliferation (31, 32). Our conclusions would support the proposal that a sustained activation of the MAPK pathway would result in a different program of transcriptional activation when compared with transient activation of the pathway. This knowledge suggests that DNA microarray technology, designed to detect alterations in transcription, rather than alterations in translation or posttranslational modification, will be an efficient tool in further understanding phenotypes dependent on a the duration of signal pathway activation.

With regard to the IFN-{gamma} pathway in particular, this work resolves why the MHC class II genes, which are completely dependent on CIITA, are not activated by low levels of IFN-{gamma} (Ref. 9 ; G.B., unpublished observations). Low levels of IFN-{gamma} can activate STAT1 and lead to a saturating level of STAT1 but do not lead to the sustained STAT1 response that is at least indirectly, and possibly directly, required to maintain CIITA expression after 24 h. It is possible that the falloff of activated STAT1, when the pathway is only transiently activated, leads to lack of CIITA promoter occupancy and a lack of CIITA promoter activation by STAT1. Alternately, a transient occupancy of the CIITA GAS may be sufficient for a sustained CIITA induction, as in the case of the hGBP promoter (30), but the falloff of STAT1 leads to cessation of IRF-1 synthesis that would be required for continued CIITA promoter activation.

Functional consequences of sustained and transient IFN-{gamma} signaling

The knowledge that the IFN-{gamma} signaling pathway can be regulated by the duration of the pathway activation raises the question of whether the recently described, short cytokine bursts from T cells (33) are ordinarily sufficient for eradicating infections while minimizing the possibility of developing HLA class II-dependent autoimmunity, which could be more likely with sustained STAT1 activation. Sustained IFN-{gamma} induction of p21 and IRF-1 transcripts is required for IFN-{gamma}-mediated growth suppression and apoptosis in ovarian cancer cells (34). Our prediction, based on the work above, is that the sustained induction of these portions of the pathway are required to transcriptionally activate genes specifically required for growth suppression and apoptosis. In addition, IFN-{gamma} induced H2O2 synthesis occurs with one round of IFN-{gamma} receptor internalization, whereas multiple rounds of IFN-{gamma} receptor recycling are required for IFN-{gamma} induced antitumoricidal activity in macrophage (35). Again, we predict that multiple rounds of receptor internalization lead to the transcriptional activation of genes that are dependent on sustained STAT1 activation and that are required for antitumoricidal activity.

Transient STAT1 activation and tumorigenesis

In retrospect, because a minimal duration of STAT1 activation is required for CIITA activation, there is no basis for expecting CIITA activation to occur in the human tumor lines described in this study (Fig. 1Go). It is possible that numerous mechanisms function in the silencing of the CIITA gene in these or other tumor lines. However, given the results that indicate that a sustained duration of STAT1 activation is required for high level CIITA activation, no significant amount of CIITA would be expressed in these lines without a higher and sustained level of STAT1 activation, regardless of other mechanisms of CIITA gene silencing in these cells. Several reports have indicated that the lack of all or part of the IFN-{gamma} signaling pathway facilitates tumorigenesis in mice (36). However, with few exceptions, it is not clear how or to what extent human tumor cells lose a functional IFN-{gamma} signaling pathway as part of tumorigenesis. In the most well understood case, the loss of Rb is associated with loss of MHC class II inducibility in cell lines in culture (37) and in Rb-defective mice (38). The loss of functional IFN-{gamma} signaling could impact on tumor Ag presentation through loss of MHC class II inducibility, leading to a reduced antitumor immune response (19, 36, 39, 40), or on the susceptibility of tumor cells to undergo apoptosis (41, 42, 43). There has also been an indication that human tumor mutations lead to a loss of IRF-2 function, which would lead to an impaired CIITA response to IFN-{gamma} (18, 44). In both the Rb and IRF-2 cases, the upstream portion of the IFN-{gamma} signaling pathway remains intact, and only a part of the downstream IFN-{gamma} signaling pathway is affected by the loss of these constitutively expressed proteins. Several reports have described naturally occurring tumor-associated mutations associated with lack of STAT1 expression and loss of the upstream portion of the IFN-{gamma} signaling pathway, suggesting that loss of the IFN-{gamma} signaling pathway is a common occurrence among tumor cells (45, 46, 47). The tumor lines described in this study maintain STAT1 expression and show loss of the upstream portion of the IFN-{gamma} signaling pathway through decreased duration of STAT1 activation in the nucleus, demonstrating an additional phenotype associated with loss of IFN-{gamma} signaling in tumor cells. The decreased duration of activated STAT1 in the tumor cells could occur through receptor-associated inhibition of STAT1 activation, altered nuclear import of STAT1, or increased nuclear STAT1 dephosphorylation. Changes associated with decreased STAT1 activation may be an advantage for tumor progression, given that prolonged STAT1 activation is related to growth arrest in malignant lymphoma cells (48, 49) and apoptosis in ovarian cancer cells (34). Further studies with fresh tumor explants are required to determine whether partial loss of the IFN-{gamma} signaling pathway is a feature of tumorigenesis, because the cell lines used in this study represent long term, immortalized tissue culture lines that have conceivably acquired phenotypes in culture. However, the results lead to the question, "Is it possible to link the transient STAT1 activation phenotype to a specific tumorigenic event?"


   Acknowledgments
 
We thank Dr. Frederick Kaye for providing the NSCLC H1734 cell line and Dr. Michelle Muncaster for providing the Weri-Rb1, Y79, and MDA468-S4-Rb1 cell lines. We also thank Dr. Jeremy Boss for providing the CIITA-specific primer sequences and the human CIITA cDNA construct and Dr. Richard Jove for providing the hSIE probe sequence and for helpful discussions concerning this work. A special thanks goes to Dr. Hongquan Zhang for help with protein concentration assays.


   Footnotes
 
1 This work was supported by grants from the American Cancer Society (RPG-98-184-01-CM) and the National Institutes of Health (R01 CA81497). Back

2 Address correspondence and reprint requests to Dr. George Blanck, Department of Biochemistry and Molecular Biology, University of South Florida, College of Medicine, MDC 7, 12901 Bruce B. Downs Boulevard, Tampa, FL 33612. Back

3 Abbreviations used in this paper: IRF-E, IFN-regulatory factor element; CIITA, class II trans-activator; IRF, IFN-regulatory factor; GAS, {gamma} activation sequence; RB, retinoblastoma tumor suppressor protein; RPA, RNase A protection assay; NSCLC, non-small cell lung carcinoma; hGBP, human guanylate binding protein. Back

Received for publication June 30, 2000. Accepted for publication October 23, 2000.


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