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(The FASEB Journal. 2001;15:383-392.)
© 2001 FASEB

Regulation of monocyte chemoattractant protein (MCP)-1 transcription by interferon-gamma (IFN-{gamma}) in human astrocytoma cells: postinduction refractory state of the gene, governed by its upstream elements

Z.-H. LUCY ZHOU1, YULONG HAN, TAO WEI, SUMER ARAS2, PRIYA CHATURVEDI3, SARAH TYLER4, M. R. SANDHYA RANI and RICHARD M. RANSOHOFF*5

Department of Neurosciences, The Lerner Research Institute, and
* Department of Neurology and The Mellen Center for Multiple Sclerosis Treatment and Research, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA

5Correspondence: Department of Neurosciences, Lerner Research Institute, NC30, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195, USA. E-mail: ransohr{at}ccf.org


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Monocyte chemoattractant protein (MCP)-1 is expressed by astrocytes in diverse inflammatory states and is a key regulator of monocyte recruitment to the central nervous system (CNS). In the current study, we addressed mechanisms by which transcription of the human MCP-1 gene (hMCP-1) was terminated, after induction by interferon (IFN)-{gamma}. Our results demonstrated that IFN-{gamma}-induced transcription of hMCP-1 was followed by a refractory state, during which hMCP-1 was resistant to restimulation by either IFN-{gamma} or heterologous activators such as TNF-{alpha}. This refractory state affected the hMCP-1 gene selectively, as other IFN-{gamma}-inducible genes remained responsive to restimulation. The IFN-{gamma}-induced hMCP-1 refractory state was governed at the transcriptional level and was sensitive to protein synthesis inhibitors, suggesting a requirement for newly expressed components. A minimal 213 base pair hMCP-1 regulatory element directed both IFN-{gamma}-mediated transcription and the subsequent refractory state. We previously demonstrated that IFN-{gamma} treatment resulted in coordinate protein occupancy in vivo of two hMCP-1 promoter elements, a gamma-activated site (GAS) and a GC-rich element. During the refractory state, IFN-{gamma} treatment failed to induce protection of either the hMCP-1 GAS element or the GC box. These results furnish insight into the expression of hMCP-1 during CNS inflammation and provide the first delineation of an IFN-{gamma}-induced transcriptional refractory state.—Zhou, Z.-H. L., Han, Y., Wei, T., Aras, S., Chaturvedi, P., Tyler, S., Rani, M. R. S., Ransohoff, R. M. Regulation of monocyte chemoattractant protein (MCP)-1 transcription by interferon-gamma (IFN-{gamma}) in human astrocytoma cells: postinduction refractory state of the gene, governed by its upstream elements.


Key Words: chemokine • gene expression • interferons • STAT factors • Sp1 transcription factor


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MONOCYTE CHEMOATTRACTANT PROTEIN 1 (MCP-1), a member of the chemokine superfamily, is implicated in immune regulation, inflammatory responses, the pathogenesis of atherosclerosis, wound healing, tissue remodeling, and modulation of tumor behavior (1 2 3 4 5 6 7) . In human monocytes, MCP-1 induces chemotaxis, calcium flux, and the respiratory burst and up-regulates adhesion molecule expression and cytokine production (2) . MCP-1 is secreted by many cell types in response to lipopolysaccharide, or cytokines including interleukin (IL) -1, IL-4, interferon {gamma} (IFN-{gamma}), and tumor necrosis factor (TNF)-{alpha} (8 , 9) .

Four closely related murine and human MCPs have been identified, all of which exhibit monocyte chemoattractant activity in vitro. The specific functions of murine MCP-1 (mMCP-1) in vivo were investigated by construction and analysis of mMCP-1-deficient mice. Given the presence of four similar MCPs, it is surprising that these studies indicated that deletion of MCP-1 abrogated or altered diverse acute and chronic immune and inflammatory processes (3 4 5 6 7) . It is uncertain whether expression patterns or structural characteristics account for the nonredundant functions exerted by mMCP-1.

At the genetic level, mMCP-1 was first characterized as JE, an immediate-early gene that was activated in quiescent NIH 3T3 fibroblasts by exposure to platelet derived growth factor (PDGF) (12) . Human MCP-1 (hMCP-1) encodes a protein that is 55% homologous to its mouse counterpart (1) . Despite the relative lack of coding sequence identity, mMCP-1 and hMCP-1 are both highly expressed in diverse pathological states, suggesting that the two proteins may represent functional orthologs. Genomic elements regulating expression of the mMCP-1 gene have been characterized: four distal elements including two nuclear factor kappa-B (NF{kappa}B) binding sites and a seven base pair (bp) element in the 3'-untranslated region of the gene were responsible for induction by various stimuli, including PDGF and TNF-{alpha} (13 , 14) .

Both mMCP-1 and hMCP-1 are produced by astrocytes of the central nervous system (CNS) in response to diverse insults, including immune-mediated, post-traumatic, and ischemic inflammatory states, as well as during HIV encephalopathy (15 16 17 18 19 20 21) . Therefore, elucidating the regulation of MCP-1 in astrocytes is critical to understanding the role of this key mediator of CNS inflammation.

We previously described the mechanism whereby IFN-{gamma} treatment induced transcription of the hMCP-1 gene in astrocytic cells (22) . For these experiments, we used CRT astrocytoma cells, which closely mimic the cytokine response characteristics of human astrocytes in primary culture (23 , 24) . Initial studies established requirement of a cis-acting gamma-activated site (GAS) and the trans-acting factor signal transducer and activator of transcription (STAT)-1{alpha} for IFN-{gamma}-induced MCP-1 transcription (22) . Analysis of the proximal promoter region revealed a GC-rich consensus binding site for the ubiquitous transcription factor, Sp1, centered ~90 bp downstream of the GAS site. In vivo genomic footprinting (IVGF) revealed that the hMCP-1 GC box was not protected until 15–30 min after IFN-{gamma} treatment, temporally coincident both with inducible methylation-resistance of the GAS site and transcriptional activation of hMCP-1 (22) . Further functional analyses of the hMCP-1 promoter by transient transfection of a series of site-directed mutants indicated a cooperative interaction between the GAS element and the GC box. Electrophoresis mobility shift assays (EMSA) showed that Sp1 was constitutively abundant in nuclear extracts of CRT cells and that levels of Sp1 were unaltered at any time point after IFN-{gamma} treatment (22) . These observations led us to propose the hypothesis that hMCP-1 induction by varied stimuli would produce changes in access to the regulatory regions of the promoter, allowing binding of inducible transcription factors (STATs, NF{kappa}B family members) to their cognate sites and recruitment of ubiquitous and constitutive Sp1 to the GC-rich element (22) .

Elegant studies of the mMCP-1 gene by Boss and colleagues confirmed and extended this hypothesis (25 26 ). Using a variety of approaches in Sp1-deficient mammalian and insect cells, these workers demonstrated that a proximal regulatory region containing the Sp1 binding site was essential for mMCP-1 transcription in response to either TNF-{alpha} or PDGF, even though the mechanisms of induction by these two agents clearly differed (25 , 26) . The absence of functional Sp1 in mammalian cells precluded in vivo assembly of transcription factor NF{kappa}B on a distal regulatory element of the mMCP-1 promoter in TNF-{alpha}-treated cells, despite the demonstration of abundant and functional NF{kappa}B in vitro in cell extracts (26) . Finally, blockade of PDGF-induced MCP-1 transcription by trans-retinoic acid was associated with defective occupancy in vivo of the Sp1 binding site and other proximal regulatory elements. Similar concepts had been developed by Collins et al. in studies of the vascular cell adhesion molecule promoter (27) .

During CNS inflammation, MCP-1 expression is strikingly transitory; this characteristic implies the presence of a negative regulatory mechanism (15) . Gene induction by IFNs, which are implicated in the pathogenesis of immune-mediated inflammatory states, is known to be transient in many cases (28) . The present study extends our characterization of IFN-{gamma} regulation of hMCP-1 expression in astrocytoma cells. Unexpectedly, we found that the hMCP-1 gene was resistant to restimulation after IFN-{gamma}-induced transcription had terminated. This resistance to restimulation (termed a refractory state in this report) was long-lasting and operated at the level of gene transcription. The initial IFN-{gamma}-induced transcription and postinduction refractory state of hMCP-1 were both mediated through a 213 bp element upstream of the structural gene, which was characterized in our previous report (22) .

During the IFN-{gamma}-mediated refractory state, hMCP-1 transcription could not be induced either by reexposure to IFN-{gamma} or by heterologous stimuli such as TNF-{alpha}. The refractory state was selective for hMCP-1, as other chemokine genes including IP-10 remained responsive to transcriptional activators, including IFN-{gamma}. Inhibition of protein synthesis during the initial exposure to IFN-{gamma} fully reversed the refractory state, suggesting dependence on newly synthesized components. Analysis of promoter occupancy in vivo during the refractory state revealed impaired IFN-{gamma}-inducible protection of the GAS site and the GC-rich element, suggesting decreased availability of the hMCP-1 promoter to factors required for gene transcription.

This is the first report of an IFN-{gamma}-inducible transcriptional refractory state, and suggests mechanisms by which MCP-1 expression in vivo may be temporally restricted. Further, these data provide additional support for the hypothesis that cytokine-regulated cis-elements and the GC box cooperate to govern transcription of both human and murine MCP-1.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
The CRT astrocytoma cell line, as described previously (24) was derived from a grade IV human astrocytoma. Experiments reported here were done between passages 10 and 40. CRT cells were routinely maintained in RPMI 1640, supplemented with 2 mM L-glutamine and 10% fetal bovine serum.

Reagents
Purified human recombinant IFN-{gamma} (1.9x107 units/mg protein) was purchased from Genentech Inc. (South San Francisco, Calif.). TNF-{alpha} was purchased from Becton-Dickinson (San Jose, Calif.). Polybrene (hexadimethrine bromide), cycloheximide, and dimethyl sulfoxide were purchased from Sigma Chemical Company (St. Louis, Mo.).

RNA isolation, Northern blot, and RNase protection analysis
Initial studies established optimal time points for determination of steady-state levels of hMCP-1 mRNA by Northern and nuclease protection analyses. Although the fold induction of MCP-1 message varied among experiments, the time course of accumulation and decay was reproducible. Total cellular RNA was isolated from 90% confluent CRT cells using TRIzol (GIBCO BRL, Grand Island, N.Y.) according to the manufacturer’s instructions; 30 µg of total RNA was denatured with formaldehyde, electrophoresed, and transferred to GeneScreen nylon membrane (Biotechnology Systems, NEN Research Products, Boston, Mass.). Hybridization was carried out at 42°C for 16 h in a solution containing denatured human MCP-1 cDNA probe (1x106 cpm/ml). A 740 bp human MCP-1 cDNA hybridization probe was generated by PstI digestion of pGEM-hJE34 (a generous gift from Dr. B.J. Rollins, Dana-Farber Cancer Institute). The gel-isolated insert was radiolabeled by random priming as described previously (22) . Autoradiograms show results typical of those obtained from three separate experiments. Subsequent analyses of hMCP-1 mRNA accumulation were performed by RNase protection, using riboprobes, as described (22) . These experiments were repeated twice and representative results are shown.

Nuclear run-on analysis
Initial studies established optimal time points for analyzing hMCP-1 gene transcription by nuclear run-on assays. For each data point, 5 x 106 cells at 70–80% confluency were washed, scraped in ice-cold phosphate-buffered saline (PBS), and pelleted. Nuclei were isolated and nascent transcripts were labeled with [32P]-UTP at 25°C for 45 min, as described previously (24) . As hybridization substrates, plasmid DNAs were denatured and spotted onto nitrocellulose membranes, which were excised with a hole punch. Hybridization with individual substrates was carried out in minimal volumes at 42°C for 3 days, with 107 cpm/ml of radiolabeled RNA probe generated from nuclei representing various experimental conditions. After high-stringency washes at 65°C, individual nitrocellulose membrane circles were mounted on filter paper supports to generate autoradiograms. For some experiments, hybridization signals were quantitated in a PhosphorImager. Transcriptional activation was calculated as the MCP-1/ß-actin densitometric ratio. These experiments were repeated twice and representative results are shown.

Promoter-reporter construction
Construction of expression plasmids containing a series of deletion and substitution mutants of the MCP-1 promoter, directing either CAT or luciferase reporters, has been described in detail (22 , 29) . GL-IP10 was generated by insertion of a 972 bp IP-10 genomic fragment into the promoterless pGL3-basic vector (30) .

Transient transfection
Polybrene with 50 µg of supercoiled test plasmid DNA was used to transfect CRT astrocytoma cells, as described (22) . As an internal control for transfection efficiency, 1 µg of a simian virus 40 promoter-ß-galactosidase reporter plasmid, pCH110 (Pharmacia, Piscataway, N.J.), was cotransfected with each test plasmid. After cells were transfected, heat-shocked, and rested overnight, they were pooled and split into 100 mm dishes to control for differential transfection efficiency.

Initial studies established optimal time points for analyzing hMCP-1 promoter-reporter expression in transient transfection assays. Using this information as a guide, cells were subjected to various protocols of cytokine stimulation, washing, and restimulation; cells were then incubated overnight in complete cytokine-free medium to allow CAT or luciferase protein to accumulate, before lysis and enzyme assay.

CAT and luciferase assays were performed using standardized protocols (22) . ß-Galactosidase activity was measured in cell lysates with the ß-galactosidase enzyme assay system kit (Promega, Madison, Wis.). Luciferase or CAT activities of cytokine-exposed or control cells were normalized to ß-galactosidase activity. Results presented in this study were obtained from three to four separate experiments. For statistical analysis, the t test was used to evaluate paired samples, with significance set at P<0.05.

IVGF
Initial studies established optimal time points for analyzing IFN-{gamma}-induced occupancy of the hMCP-1 promoter by IVGF. In vivo methylation of cellular DNA and DNA preparation were performed as described (14 , 22 , 30) . Ligation-mediated polymerase chain reaction was carried out according to the procedure of Mueller et al. (31) , as adapted for mMCP-1 by Ping et al. and with modifications for hMCP-1, as we previously described (22 , 30) . Both strands of the 213 bp promoter proximal region of the hMCP-1 gene were analyzed. This analysis was repeated twice; representative results for the noncoding strand (the site of asymmetric Sp1 binding to the GC-rich element) are shown. The annealing temperatures for the coding strand primers were 59°C, 66°C, and 69°C. Coding strand primers were:

5'-TGTGGTTCAAGGAGAAGAAGAGGG-3'

5'-GCTATGAGCAGCAGGCAC-AGAAGG-3'

5'-CAGGCACAGAAGGGCGGCAGAGAC-3'.

The annealing temperatures for noncoding strand primers were 59°C, 66°C, and 69°C. Primers for the noncoding strand were:

5'-CCCTCTTAGTTCACATCTGTGGTCAG-3'

5'-CCCATCCTCCCCATTTGCTCATT-3'

5'-TCCCCATTTGCTCATTTGGTCTCAGCAG-3'.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
After induction with IFN-{gamma}, the hMCP-1 gene is refractory to restimulation
As shown in Fig. 1 , hMCP-1 mRNA accumulated rapidly after IFN-{gamma} treatment reached a maximum by 8 h and decayed markedly by 24 h in the continued presence of IFN-{gamma}.



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  		Figure 1. Time course of hMCP-1 mRNA accumulation induced by IFN-{gamma}. CRT cells were treated for the indicated times with IFN-{gamma} (100 U/ml); mRNA levels were determined by Northern blot analysis and results quantitated on a PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.), with hMCP-1/ß-actin ratios shown in the bar histogram. The results shown are representative of three separate experiments.

To determine (Fig. 2 ) whether this down-regulation was associated with postinduction repression of hMCP-1 mRNA expression, we incubated CRT cells with IFN-{gamma} for 24 h, terminated IFN-{gamma} stimulation by extensive washing, incubated in IFN-{gamma}-free media for varying times, and restimulated with IFN-{gamma}. Unexpectedly, hMCP-1 was resistant to re-induction by IFN-{gamma} after down-regulation (Fig. 2 , lanes 4–6 vs. lane 2). This refractory state was equally robust after 4, 8, or 24 h of IFN-{gamma}-free incubation (Fig. 2 , lanes 4–6). Thus, IFN-{gamma}-mediated induction of MCP-1 was succeeded by a refractory state during which the gene was resistant to re-induction with IFN-{gamma}. In parallel studies, the IP-10 gene remained responsive to reinduction with IFN-{gamma} (Fig. 2 , lanes 4–6), indicating that IFN-{gamma} receptor down-regulation or transcription factor exhaustion was not responsible for the inability of restimulation with IFN-{gamma} to induce hMCP-1 mRNA accumulation.



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  		Figure 2. Refractory state of hMCP-1 but not IP-10 after induction with IFN-{gamma}. CRT cells were treated with IFN-{gamma} (100 U/ml) for 4 h (lane 2) or 24 h (lane 3) before Northern analysis, demonstrating down-regulation of hMCP-1 but not IP-10 mRNA after 24 h exposure to IFN-{gamma}. The refractory state of hMCP-1 is shown in lanes 4–6: after 24 h treatment with IFN-{gamma}, cells were washed five times with PBS and incubated in cytokine-free medium for 4 h (lane 4), 8 h (lane 5), or 24 h (lane 6). Cells were then restimulated with IFN-{gamma} for 4 h (lanes 4–6). hMCP-1, IP-10, and ß-actin mRNAs were analyzed by Northern blotting, and quantitated on a PhosphorImager; bar histograms indicate chemokine/ß-actin ratios. Representative results of three independent experiments are shown.

After induction with IFN-{gamma}, the hMCP-1 gene is resistant to restimulation with TNF-{alpha}
TNF-{alpha} is a potent and well-characterized stimulus for MCP-1 transcription. We asked whether the hMCP-1 gene was resistant to induction with TNF-{alpha} during the IFN-{gamma}-induced refractory state. After treatment for 4 h with either IFN-{gamma} or TNF-{alpha} (Fig. 3 , lanes 2, 5), CRT cells accumulated abundant hMCP-1 mRNA. When cells were pretreated with IFN-{gamma} for 4 h, washed extensively, incubated in cytokine-free medium for 4 h, and reexposed either to IFN-{gamma} or TNF-{alpha}, MCP-1 mRNA was not induced above baseline levels (Fig. 3 , compare lane 1 with lanes 3 and 4). This result indicated that the IFN-{gamma}-mediated postinduction refractory state rendered the hMCP-1 gene resistant to induction by TNF-{alpha}, an efficient heterologous stimulus.



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  		Figure 3. During the IFN-{gamma}-mediated refractory state, hMCP-1 is resistant to induction by TNF-{alpha}. During the first treatment, as indicated, CRT cells were incubated in cytokine-free medium (lanes 1, 2, 5) or induced with IFN-{gamma} (100 U/ml; lanes 3 and 4) for 4 h. Cells were then extensively washed and incubated for 4 h during the second treatment, as indicated, with medium (lane 1), IFN-{gamma} (lanes 2, 3), or TNF-{alpha} (lanes 4, 5) before preparation of total cellular RNA and analysis of MCP-1 and {gamma}-actin accumulation by nuclease protection assay. Representative results from two separate experiments are shown. After exposure to IFN-{gamma} during the first treatment (lane 4), CRT cells were resistant to induction of hMCP-1 by TNF-{alpha}.

The refractory state of the MCP-1 gene occurs at the level of transcription
To determine whether the IFN-{gamma}-induced refractory state of the hMCP-1 gene was governed at the transcriptional level, nuclear run-on experiments were conducted. In IFN-{gamma}-treated CRT cells, hMCP-1 transcription was induced at 2 h and declined markedly 1 h later (Fig. 4 : compare 2 h and 3 h). Restimulation of cells with IFN-{gamma} failed to recover transcription of the hMCP-1 gene (Fig. 4 : 3 h+rest+restim).



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  		Figure 4. The IFN-{gamma}-induced refractory state occurs at the level of transcription and affects hMCP-1 but not IP-10. Transcription rates of the hMCP-1, IP-10, and ß-actin genes were analyzed by evaluation of nascent transcripts, after isolation of nuclei from cells exposed to IFN-{gamma} (500 U/ml) for varying times. For restimulation, cells were exposed for 3 h to IFN-{gamma}, washed, incubated in cytokine-free medium for 1 h, and exposed IFN-{gamma} for 2 h. Densitometric signals were quantitated on a PhosphorImager and chemokine gene transcription was normalized to ß-actin. Representative data from one of two experiments are shown.

The transcriptional refractory state of the MCP-1 gene was selective, as the IP-10 gene remained fully responsive to IFN-{gamma} restimulation (Fig. 4) . The transcription factor STAT-1{alpha} is required for the IFN-{gamma}-inducible expression of both hMCP-1 and IP-10 in CRT cells (22 , 30 , 32) . Therefore, these data indicated that post-IFN-{gamma} receptor events, including activation of the STAT-1{alpha} transcription factor, were maintained during the refractory state and that their failure did not account for the unresponsive state of the hMCP-1 gene.

The IFN-{gamma}-induced refractory state of the hMCP-1 gene is directed by a 213 bp element upstream of the structural gene
We previously characterized a 213 bp element of the hMCP-1 gene that directed the transcriptional response to IFN-{gamma} in CRT cells (22) . To test whether the IFN-{gamma}-induced refractory state was governed by upstream elements of the hMCP-1 gene, we modified transient transfection assays, using hMCP-1 promoter-reporter constructs documented in previous studies. The premise of these experiments was that repetitive cycles of cytokine stimulation should produce additive accumulation of CAT or luciferase activity if the promoter remained responsive to restimulation. Therefore, experiments were performed to determine whether repeated cycles of stimulation would result in additive accumulation of hMCP-1 promoter-reporter CAT or luciferase activity.

We tested this assay, using a previously characterized IP-10 promoter-reporter (GL-IP-10) (30) . Luciferase activity was strongly induced by IFN-{gamma} (100 U/ml) treatment (Fig. 5A , 6 h). After extensive washing, incubation for 3 h in cytokine-free medium, and IFN-{gamma} (100 U/ml) restimulation, GL-IP-10 reporter activity accumulated in a nearly additive fashion (Fig. 5A , compare 6 h with 6 h+Restim), indicating that the reporter protein remained stable in IFN-{gamma}-treated CRT cells during the time frame of the experiment. This result indicated that a transcriptional refractory state could be detected as a failure of reporter activity to accumulate in additive fashion after repetitive cycles of cytokine stimulation.



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  		Figure 5. The hMCP-1 refractory state is mediated by 213-bp of sequence upstream of the structural gene. A) IP-10 promoter-reporters do not become refractory to restimulation after induction by IFN-{gamma}. CRT cells were transfected with pGL-IP-10 as described in Materials and Methods. Cells were either untreated (0) or treated with 500 U/ml of IFN-{gamma} for 6 h (6 h), followed by extensive washing, 3 h incubation without cytokine, and 2 h of restimulation (6 h+restim). Representative data from one of two experiments are shown. B) hMCP-1 promoter-reporters exhibit an IFN-{gamma}-inducible refractory state. Each promoter-reporter is designated according to the amount in bp of hMCP-1 upstream sequence, as previously reported (22) . Transiently transfected CRT cells (in 15 cm2 dishes) were treated as indicated. Lanes 1: mock-treated-controls; lanes 2: IFN-{gamma} for 2 h without restimulation; lanes 3: IFN-{gamma} for 2 h, 3 h release from cytokine, and 2 h restimulation with IFN-{gamma}. CAT assay (normalized to ß-galactosidase activity) was performed as described in Materials and Methods. Results were obtained from three to four separate experiments. The y-axis shows fold stimulation relative to control, which is set at one. This display allows for comparison among several different promoter-reporters. Typical percent acetylation in these experiments was 5–10%. Means ± standard deviations are shown.

Serial deletion constructs containing various length of 5'-flanking sequences of the hMCP-1 promoter were inserted upstream of CAT reporter gene, reserved as controls (Fig. 5B , lanes 1 for each construct), or induced (Fig. 5B , lanes 2) with IFN-{gamma} (100 U/ml; 2 h). Significantly increased CAT activity was induced by IFN-{gamma} (P<0.001; t test) from constructs containing 3.5 kb, 394 bp, 292 bp, or 213 bp of the hMCP-1 promoter, whereas a 141 bp hMCP-1 promoter-reporter construct failed to respond to stimulation (Fig. 5B , compare lanes 1 and 2). These results reproduced data from our previous report (22) indicating that the 213 bp construct contains a minimal IFN-{gamma}-inducible hMCP-1 promoter.

To define the hMCP-1 promoter element that governed the refractory state, CRT cells transfected with these serial deletion constructs were treated with IFN-{gamma} for 2 h, washed extensively, incubated without IFN-{gamma} for 3 h, and then reexposed to IFN-{gamma} for an additional 2 h before normalized CAT assay (Fig. 5B , lanes 3). The IFN-{gamma}-inducible hMCP-1 promoter-reporters were uniformly refractory to restimulation (Fig. 5B , compare lanes 2 and 3; for each comparison, P>0.5). Experiments with hMCP-1 promoter-luciferase/reporter constructs provided consistent results (not shown). Taken together, these results indicated that 213 bp of hMCP-1 upstream sequence dictated the response to IFN-{gamma}, including both the initial induction and the subsequent refractory state. These results also provided a separate line of evidence that the IFN-{gamma}-inducible refractory state of the hMCP-1 gene was governed at the transcriptional level.

The IFN-{gamma}-induced refractory state of the hMCP-1 gene is sensitive to cycloheximide
The IFN-{gamma}-induced MCP-1 refractory state could be mediated by an active process, dependent on de novo or ongoing protein synthesis. To address this possibility, the refractory state was tested for sensitivity to protein synthesis inhibition with cycloheximide (CHX). Assays included Northern analysis of steady-state mRNA accumulation (Fig. 6 ), nuclear run-ons to evaluate transcriptional activity of the hMCP-1 gene (Fig. 7 ), and transient transfection analysis (Fig. 8 ). When CHX was included in the initial IFN-{gamma} treatment of CRT cells, the refractory state was reversed as determined by all three assays.



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  		Figure 6. CHX treatment during IFN-{gamma} stimulation abolishes the subsequent refractory state of the hMCP-1 gene. CRT cells were reserved as controls (lane 1) or treated for 4 h (lane 2) with IFN-{gamma} (100 U/ml) to demonstrate induction of hMCP-1 or IP-10 mRNA, as assayed by Northern blotting. To analyze the refractory state (lanes 3 and 5), CRT cells were treated with IFN-{gamma} (100 U/ml) for 12 h, extensively washed and incubated in cytokine-free medium for 1 h, and retreated for 4 h with IFN-{gamma} before analysis of chemokine and ß-actin mRNAs. With the initial 12 h incubation, CHX (50 µg/ml) was either included (lane 5) or omitted (lane 3). Lane 4: as a viability control for exposure to CHX, CRT cells were incubated with CHX alone (50 µg/ml) for 12 h before washing, rest for 1 h, treatment for 4 h with IFN-{gamma} (100 U/ml), and Northern analysis.



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  		Figure 7. CHX treatment during IFN-{gamma} stimulation prolongs transcription of the hMCP-1, but not the IP-10, gene. CRT cells were treated with IFN-{gamma} (500 U/ml) for 2 or 4 h, as indicated, with or without CHX (50 µg/ml). Autoradiograms demonstrating transcription of hMCP-1, IP-10, and ß-actin were generated on a PhosphorImager. pBS (pBluescript; Promega) vector: negative control for nonspecific hybridization. Transcription of hMCP-1 but not IP-10 was markedly prolonged at both 2 h and 4 h by inclusion of CHX with IFN-{gamma}. CHX alone did not stimulate transcription of hMCP-1 (not shown).



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  		Figure 8. CHX treatment during IFN-{gamma} stimulation abrogates resistance of a minimal hMCP-1 promoter-reporter to IFN-{gamma} restimulation. CRT cells were transfected with the 213 bp hMCP-1 promoter-reporter, as described in Materials and Methods; cells were then incubated in the presence or absence of IFN-{gamma} with or without CHX, as indicated, before incubation overnight in medium, lysis, and normalized assay of CAT reporter expression. Control: mock-treated; stim: treated with IFN-{gamma} for 2 h; stim-re/stim: 2 h of IFN-{gamma}, extensive washing; 3 h of cytokine-free incubation and 2 h of restimulation with IFN-{gamma}; stim-re/stim-CHX: 2 h of CHX with IFN-{gamma}, extensive washing; 3 h of cytokine-free incubation and 2 h of restimulation with IFN-{gamma} alone; CHX: cycloheximide alone for 2 h; stim+CHX: treatment with CHX and IFN-{gamma} for 2 h. The y-axis shows fold stimulation relative to control, which is set at one.

For Northern analysis of steady-state levels of hMCP-1 mRNA, CRT cells were exposed to IFN-{gamma} for 12 h in the presence (Fig. 6 , lane 5) or absence (Fig. 6 , lane 3) of CHX, washed, incubated in fresh medium for 4 h, and retreated with IFN-{gamma} for 4 h. The refractory state was entirely reversed by inclusion of CHX with IFN-{gamma} during the initial induction (Fig. 6 , compare lanes 3 and 5). In parallel assays of mRNA for IP-10, another chemokine gene highly expressed in astrocytes (33) , the refractory state (Fig. 6 , compare lanes 3 and 5) was not observed. Pretreatment of CRT cells for 12 h with CHX alone before washing, cytokine-free incubation, and restimulation (Fig. 6 , lane 4) excluded gross effects of this treatment on cell viability.

Nuclear run-on experiments indicated that inclusion of CHX with IFN-{gamma} prolonged hMCP-1 gene transcription (Fig. 7 , compare IFN-{gamma}-induced hMCP-1 transcription after 2 h and 4 h in the presence or absence CHX). This observation suggested that the refractory state of the hMCP-1 gene was established during down-regulation of IFN-{gamma}-induced transcription. Therefore, CHX treatment both lengthened the period of active IFN-{gamma}-induced hMCP-1 gene transcription (Fig. 7) and abrogated the refractory state (Fig. 6) . These results are compatible with those previously reported by Larner and colleagues, who described IFN-ß-mediated regulation of two genes. These workers reported that induction of these two genes by IFN-ß was followed by a dramatic transcriptional down-regulation, which was abrogated by CHX, suggesting an active mechanism of suppression (34) . Exposure to CHX did not extend, but moderately impaired, IFN-{gamma}-induced transcription of the IP-10 gene (Fig. 7) , further indicating selective regulation of hMCP-1 by IFN-{gamma}.

Transient transfection experiments (Fig. 5B ) demonstrated that the refractory state of the hMCP-1 gene was governed by upstream elements of the gene. This assay was also used to address the dependence of the refractory state on ongoing protein synthesis. As previously observed, repetitive cycles of stimulation with IFN-{gamma} failed to induce CAT reporter activity above levels obtained after individual cycles of stimulation (Fig. 8 , compare ‘stim’ with stim-re/stim). However, when CHX was included with IFN-{gamma} during the initial induction (stim-re/stim-CHX), accumulation of CAT activity increased markedly after a second cycle of stimulation with IFN-{gamma} (Fig. 8 , compare stim-re/stim with stim-re/stim-CHX). CHX alone did not induce expression of the promoter-reporter (Fig. 8 , compare control with CHX).

Addition of CHX to IFN-{gamma} did not augment induction of the promoter-reporter (Fig. 8 , compare stim with stim-CHX). This result was compatible with observations shown in Figs. 6 and 7 in which CHX prolonged transcription of the hMCP-1 gene, but did not markedly increase mRNA accumulation. Together, observations described in Figs. 6 7 8 indicated that the IFN-{gamma}-induced transcriptional refractory state of the hMCP-1 gene was reversed entirely by inhibiting protein synthesis with CHX.

In vivo state of the IFN-{gamma}-regulated MCP-1 promoter
Our previous analysis showed that IFN-{gamma}-mediated occupancy of the upstream GAS and GC-rich elements of the hMCP-1 gene occurred coincident with hMCP-1 gene transcription (22) . Functional assays of site-directed mutants of the GAS site and GC box demonstrated the critical importance of both elements for efficient induction of hMCP-1 gene transcription by IFN-{gamma} (22) . To determine the in vivo occupancy of these elements during the IFN-{gamma}-induced transcriptional refractory state, genomic footprinting analyses of the hMCP-1 promoter were conducted (Fig. 9 ). CRT cells were treated for 15 min with IFN-{gamma} and immediately assayed (during gene induction) or washed and incubated in IFN-{gamma}-free medium for 3 h before retreatment with IFN-{gamma} (during the refractory state).



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  		Figure 9. Altered hMCP-1 promoter occupancy during the IFN-{gamma}-induced refractory state. A) Occupancy of the hMCP-1 GAS element. The pattern of methylation resistance and hypersensitivity in the region of the GAS site of the MCP-1 promoter (noncoding strand) is shown. Lane assignment and symbol representation are indicated in the legend of panel B. Two GAS element methylation-resistant residues are indicated (arrows). B) Occupancy of the hMCP-1 GC box. CRT cells were mock-treated (lane 2) or exposed to IFN-{gamma} (500 U/ml) for 15 min (lane 3); treated with IFN-{gamma} for 15 min, followed by extensive washing, 3 h of cytokine-free incubation, and 15 min of restimulation (lane 4); or treated with IFN-{gamma} for 5 h (lane 5). Genomic DNA was prepared and analyzed by IVGF as described in Materials and Methods. Lane 1 shows analysis of DNA methylated in vitro. The region of the GC box on the noncoding strand is shown in the autoradiogram, with numbering by reference to the transcriptional start site. Arrow indicates an IFN-{gamma}-inducible methylation-resistant guanine residue.

After 15 min of IFN-{gamma} treatment (Fig. 9A , lane 3), strong protection of the core residues of the GAS site—G-209 and G-210— was observed (arrows), along with methylation hypersensitivity of flanking residue G-201 and weak protection of G-200. Residues outside the regulatory element (G-154 and nearby G residues) were unaffected. After 5 h, the pattern of methylation resistance of these GAS residues returned to that observed in mock-treated cells (Fig. 9A , compare lanes 2 and 5). During the refractory state, IFN-{gamma}-induced occupancy at the GAS element of the hMCP-1 promoter was markedly altered, and resembled the pattern observed in mock-treated cells or after the termination of transcription after 5 h of IFN-{gamma} (Fig. 9A ). In particular, protection at G-209, G-210, and G-200 was not induced by IFN-{gamma} treatment during the refractory state (Fig. 9A , lane 4). Residue G-201 exhibited methylation hypersensitivity during the refractory state, suggesting incomplete assembly of transcription factors on the refractory hMCP-1 promoter. Concurrently, after 15 min of IFN-{gamma} treatment (Fig. 9B , lane 3) the GC box of the noncoding strand became DMS resistant, most evident at G-123 and nearby residue G-140 (arrowhead). Located between the two regulatory elements, residue G-154 was unaffected by cytokine treatment at any time (Fig. 9A , B ). During the refractory period, IFN-{gamma} treatment failed to induce altered methylation sensitivity in the extended GC box (Fig. 9B , compare lanes 3 and 4). These results suggested that the IFN-{gamma}-induced refractory state of the hMCP-1 gene in CRT astrocytoma cells was associated with impaired access of critical transcription factors to regulatory elements of the gene.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we provide evidence that IFN-{gamma}-induced transcription of the hMCP-1 gene in CRT astrocytoma cells is succeeded by a refractory state of the gene, a novel phenomenon for IFN-{gamma}-inducible genes. Further, this postinduction repression is mediated through upstream elements of the hMCP-1 gene and depends on an active process. Our conclusions are based on the following observations: 1) treatment of CRT cells with IFN-{gamma} resulted in a rapid, transient accumulation of hMCP-1 mRNA, governed at the level of transcription; 2) after induction by IFN-{gamma}, the hMCP-1 gene (but not other IFN-{gamma}-responsive genes) became resistant to restimulation with either IFN-{gamma} or TNF-{alpha}; 3) the refractory state was abrogated by exposure to CHX during the initial induction with IFN-{gamma}.

The phenomenon of a postinduction transcriptional refractory state has been well established, and several mechanisms, including inducible repressors, have been postulated (34 , 35) . Maniatis et al. examined virus-mediated induction and postinduction repression of the IFN-ß gene. It was convincingly demonstrated that a virus-inducible cellular component termed PRDI-BFI acted as a postinduction repressor by binding directly to positive regulatory elements of the IFN-ß promoter (36 37 38) . Larner and Darnell showed that the transcriptional response of two IFN-ß-inducible genes was followed by a potent refractory state, which required de novo or ongoing protein synthesis (34) . Our studies also suggested that the refractory state of the hMCP-1 gene was dependent on newly expressed proteins. In particular, CHX completely reversed IFN-{gamma}-induced resistance to restimulation (Fig. 6) and prolonged the duration of hMCP-1 transcription (Fig. 7) . These results are thoroughly compatible with those reported by Larner and colleagues in their studies of postinduction repression of IFN-ß-inducible genes (34) . Consistent with this interpretation, CHX treatment also abolished the IFN-{gamma}-induced resistance of hMCP-1 promoter-reporters to restimulation (Fig. 8) .

In vivo genomic footprinting (IVGF) proved useful to analyze the refractory state of the hMCP-1 promoter. This analysis was performed after cells were treated for 15 min with IFN-{gamma} (Fig. 9 , lanes 3) or treated, rested, and restimulated with IFN-{gamma} for 15 min (Fig. 9 , lanes 4) during the refractory state. The results indicated substantial differences in hMCP-1 promoter occupancy in cells permissive for transcription (Fig. 9 , lanes 3) as compared with cells expressing the refractory state (Fig. 9 , lanes 4), suggesting that impaired access of the promoter to STAT-1{alpha} homodimers (at the GAS site) and Sp1 (at the GC box) rendered the promoter transcriptionally inactive (22) .

Recent work by Boss and colleagues characterized the transcriptional response of the mMCP-1 gene to varied stimuli, including PDGF and TNF-{alpha} (25 26) . Our current results demonstrate strikingly similar regulation of hMCP-1 and mMCP-1. In both cases, inducible and constitutive transcription factors are readily detected in cell extracts by EMSA yet fail to associate in vivo with MCP-1 promoters unless conditions are permissive for gene transcription. Further, the present report supports the critical involvement of the GC box for expression of the hMCP-1 gene, an attribute documented by Boss et al. for mMCP-1.

We considered the possible involvement of previously described IFN-{gamma}-inducible inhibitory trans-acting factors such as interferon regulatory factor 2 (IRF-2) or ICSBP. Sequence analysis of the 213 bp hMCP-1 regulatory region revealed an IRF binding motif near the transcription start site, but no binding activity was detected by EMSA when using a probe derived from this region (not shown).

As shown in our previous studies, STAT-1{alpha} was essential for IFN-{gamma} induction of the hMCP-1 gene. Therefore, the recently described suppressors of cytokine signaling (SOCS) proteins (39 40 41) were potential mediators of the specific down-regulation and refractory state of hMCP-1. However, intact expression of STAT-dependent genes such as IP-10 during the refractory state excluded this possibility, as SOCS act upstream of tyrosine phosphorylation, nuclear translocation, and DNA binding by STATs (39) . For similar reasons, the inducible (42) Janus kinase inhibitors would also appear to be unlikely candidates to explain our findings in these studies.

In summary, this report documents a selective refractory state of hMCP-1, an IFN-{gamma}-inducible gene. The down-regulation and refractory state of the hMCP-1 gene indicate the presence of a gene-specific inducible negative feedback mechanism that operates at the transcriptional level. Elucidating this refractory state will provide useful information about how hMCP-1, a key regulatory factor for CNS inflammation, is governed in physiology and disease.


   ACKNOWLEDGMENTS
 
This study was supported by NIH grants 2RO1-NS32151, 2PO1-CA62220 and National Multiple Sclerosis Society grant RG2362 to R.M.R. and by the Williams Family Multiple Sclerosis Research Fund.


   FOOTNOTES
 
1 Current address: Department of Neurology, Stanford University, 3801 Miranda Ave., Palo Alto, CA, USA.

2 Current address: Science Faculty, Ankara University, Ankara, Turkey.

3 Current address: Department of Oncology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406, USA.

4 Current address: Princeton University, Princeton, NJ, USA.

Received for publication May 26, 2000. Revision received July 14, 2000.
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 DISCUSSION
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