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J Biol Chem, Vol. 273, Issue 24, 14731-14737, June 12, 1998


IRT-1, a Novel Interferon-gamma -responsive Transcript Encoding a Growth-suppressing Basic Leucine Zipper Protein*

Michael V. AutieriDagger and Neera Agrawal

From the Department of Molecular Biology, Deborah Heart and Lung Research Institute, Browns Mills, New Jersey 08015

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Interferon-gamma (IFN-gamma ) inhibits proliferation of vascular smooth muscle cells (VSMCs) in culture and reduces arterial restenosis post-balloon angioplasty. The identification and characterization of IFN-gamma -specific transcripts in VSMCs are an important approach to discern the molecular mechanisms underlying vascular proliferative disease. In this report, we describe IRT-1, a novel mRNA transcript constitutively expressed in a number of human tissues, but expressed in human VSMCs only when they are stimulated with IFN-gamma . This mRNA expression is induced >200-fold 72 h after IFN-gamma treatment. IRT-1 mRNA is also acutely expressed in rat carotid arteries that are injured by balloon angioplasty. The IRT-1 cDNA transcript encodes a basic protein that contains a leucine zipper motif, a core nuclear localization sequence, and a single strongly hydrophobic region. Constitutive IRT-1 mRNA expression in human peripheral blood lymphocytes is reduced when these cells are stimulated to proliferate. Overexpression of IRT-1 protein in VSMCs alters their morphology and dramatically reduces their proliferative capacity. This study suggests that IRT-1 is an IFN-gamma -inducible factor that may regulate the progression of vascular proliferative diseases.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Vascular disease, the principal cause of heart attack, stroke, and circulatory deficit disorders, is responsible for 50% of all mortality in the western world. The use of percutaneous transluminal coronary angioplasty and stenting to treat coronary artery disease has increased exponentially in the past decade. However, the long-term efficacy of these procedures is significantly limited by the high incidence of vascular restenosis observed in as many as 40% of patients undergoing this procedure (1). The lack of a correlation between the efficacy of pharmacological interventions in preclinical and clinical studies is indicative of our poor understanding of the precise molecular mechanisms underlying this disease.

The resultant neointima formation associated with balloon angioplasty is a complex process actively involving various cell types that secrete many different cytokines and growth factors seminal to the local inflammatory response (2). These cytokines include, but are not limited to, interleukin-1, platelet-derived growth factor, and a number of colony-stimulating factors and interferons (IFNs)1 (3, 4). The major cellular component of the atherosclerotic lesion is the vascular smooth muscle cell (VSMC), which, upon exposure to these soluble factors, migrates into the intimal layer and proliferates. In restenotic lesions, VSMCs express a synthetic phenotype and secrete many cytokines and matrix proteins, which further promotes VSMC growth in an autocrine fashion (5, 6). It has been suggested that cytokine-induced activation of VSMCs in media resulting in intimal thickening is the most critical cellular event in the restenotic process (5-8).

Upon interaction with its target cell, interferons induce expression of a number of IFN-specific genes (9), which manifest their biological activities by antiviral, immune modulatory, and antiproliferative effects (10). This is particularly true in VSMCs, as it has been shown that proliferation of these cells is inhibited by lymphocyte-specific factors, primarily IFN-gamma (11, 12). The antiproliferative effects of IFN-gamma on VSMCs can be exerted indirectly, though generation of nitric oxide (13), or directly, though generation of the interferon regulatory factor (IRF) family of transcriptional regulators, which can act as activators or repressors of IFN-gamma -inducible genes (14-16). IFN-gamma is also directly antiproliferative to VSMCs in tissue culture, and the addition of IFN-gamma to proliferating VSMCs results in a reduction of c-myc expression within 2 h (17). Other data suggest that IFN-gamma inhibits VSMC proliferation by blocking the transition from G0 to G1 (18). It has also been shown that key cell cycle regulatory proteins, such as Cdc2, Cdk2, cyclins A and D, and Wee1, are also down-regulated or altered (19) by IFN-gamma treatment. Finally, VSMCs co-cultured with endothelial cells transduced with IFN-gamma cDNA grew 30-70% slower than control cells (20).

Immune cells are present in the atherosclerotic lesion and appear in greater numbers immediately following balloon angioplasty-induced vascular injury (21). IFN-gamma is produced in vivo by activated T lymphocytes, and a number of investigators have determined that T lymphocytes exert phenotypic and proliferative effects on VSMCs (12, 22). Furthermore, several studies have shown that in rats, IFN-gamma treatment inhibits arterial restenosis due to balloon angioplasty (24, 25), which is likely due to its antiproliferative effects on VSMCs. These findings have raised the possibility that IFN-gamma may represent an antirestenotic cytokine therapy. Because interferon-gamma inhibits proliferation of VSMCs in culture and IFN-gamma inhibits arterial restenosis post-balloon angioplasty, identification and characterization of IFN-gamma -specific transcripts in VSMCs are a promising strategy to discern the molecular mechanisms underlying vascular proliferative disease.

This study describes the identification and characterization of IRT-1 (interferon-responsive transcript-1), which was identified as an aberrant PCR product using gene-specific primers and RNA extracted from VSMCs stimulated with various cytokines as a template (26). IRT-1 mRNA is expressed in VSMCs only by IFN-gamma and encodes a novel basic leucine zipper (bZIP) protein. IRT-1 expression is also induced by balloon angioplasty in rat carotid arteries. Overexpression of IRT-1 in human VSMCs results in altered morphology and inhibition of cell growth. Taken together with data indicating that IFN-gamma is antiproliferative to VSMCs and exerts protective effects on rat carotid artery balloon angioplasty, modulation of IRT-1 expression may represent an important event in the regulation of VSMC growth.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cells and Culture-- VSMCs were obtained as a cryopreserved secondary culture from Clonetics Corp. (San Diego, CA) and subcultured in growth medium as described previously (26). The growth medium was changed every other day until cells approached confluence. Cells from passages 5-9 were used in the described studies. Preconfluent VSMCs were serum-starved for 48 h in Dubecco's minimal essential medium and then exposed for 20 h to 10% fetal calf serum, 10 ng/ml basic fibroblast growth factor, 100 units/ml IFN-gamma , 20 ng/ml interleukin-1beta , 20 ng/ml platelet-derived growth factor, or 2 ng/ml transforming growth factor-beta , at which time samples were processed for RNA isolation. Some samples remained untreated and were used as controls. Platelet-derived growth factor, basic fibroblast growth factor, IFN-gamma , and transforming growth factor-beta were purchased from Life Technologies, Inc.; interleukin-1beta was purchased from Boehringer Mannheim. Human peripheral blood lymphocytes (PBLs) were isolated by venipuncture from normal adult donors, isolated by Ficoll-Hypaque density gradient centrifugation, cultured in Dulbecco's minimal essential medium/complete (100 units/ml penicillin, 100 mg/ml streptomycin, 4 mM glutamine, 10% heat-inactivated fetal calf serum) plus phytohemagglutinin A (PHA) (5.0 µg/ml; Amersham Pharmacia Biotech) for the times indicated, and processed for RNA isolation.

5'-Rapid Amplification of cDNA Ends Analysis-- Total RNA was isolated from IFN-gamma -stimulated human VSMCs as described above and reverse-transcribed using oligo(dT) primer and Superscript II (Life Technologies, Inc.) according to the manufacturer's protocol. Transcripts were poly(C)-tailed with terminal deoxytransferase, and 5'-cDNA was amplified by PCR of dC-tailed cDNA using nested IRT-1-specific reverse primers. PCR products were isolated from agarose gels by glass extraction and cloned into the pCRII plasmid (Invitrogen) for DNA sequence analysis.

DNA Sequencing and Sequence Analysis-- The cDNA clone obtained above was dideoxynucleotide-sequenced on both strands in its entirety (Sequenase, U. S. Biochemical Corp.) as described previously (26). DNA and protein sequences were analyzed using the MacVector software package (International Biotechnologies, Inc.). Searches for sequence similarity were performed using the GenBankTM nucleic acid data base and Prosite protein data base through the Genetics Computer Group FASTA, BLAST, PROSITE, and PSORT programs.

Rat Left Common Carotid Artery Balloon Angioplasty-- Left common carotid artery balloon angioplasty was performed on 350-g male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) under sodium pentobarbital anesthesia (65 mg/kg intraperitoneally; Steris Laboratories, Phoenix, AZ) as described previously (26). Briefly, the left external carotid artery was cleared of adherent tissue, allowing the insertion of a 2-F Fogarty arterial embolectomy catheter (Model 12-060-2F, Baxter Healthcare, Santa Ana, CA). The catheter was guided a fixed distance down the common carotid artery to the aortic arch, inflated with a fixed volume of fluid, and withdrawn back to the site of insertion a total of three times. Once completed, the catheter was removed, and the wound was closed (9-mm Autoclips, Clay Adams) and swabbed with Povadyne surgical scrub (7.5% povidone-iodine, Chaston, Dayville, CT). Animals were housed in Plexiglas cages under a 12-h light/dark cycle with access to standard laboratory chow and drinking water ad libitum until required for tissue collection.

To isolate the carotid arteries, rats were exsanguinated via the vena cava under barbiturate anesthesia (100 mg/kg intraperitoneally). Left common carotid arteries were rapidly cleared of adherent tissue in situ, isolated, and placed directly in guanidine thiocyanate (Promega). These vessels were then immediately processed for RNA isolation. For subsequent Northern analysis, tissues were isolated from naive animals (control) and from animals that had undergone angioplasty 1, 3, and 7 days prior, and RNA was extracted as described below. Northern analysis was also performed on sham vessels (data not shown). All surgical procedures were performed in accordance with the guidelines of the Animal Care and Use Committee of Deborah Research Institute and the American Association for Laboratory Animal Care.

RNA Isolation and Northern Blot Analysis-- For each time point studied, four or five left carotid arteries were pooled, or VSMCs from culture were isolated, and total RNA was obtained by standard techniques as described (26). Equal amounts of RNA were loaded and separated on a formaldehyde-containing 1.3% agarose gel, transferred to nitrocellulose, and hybridized (0.25 M NaCl, 1% SDS, 50% formamide, 2× Denhardt's solution, 25 µg of denatured salmon sperm DNA, and 5% dextran sulfate at 42 °C overnight) with the indicated probe. All probes were alpha -32P-labeled by the random priming method (Boehringer Mannheim) (all isotopes were from Amersham Pharmacia Biotech). Blots were washed under high stringency (0.2× sodium citrate and 0.1% SDS at 65 °C) and exposed to film for 6-48 h at -80 °C. The same filter was stripped and subsequently hybridized with the various DNA probes. The beta -actin probe was generated from PCR amplimers (CLONTECH). Relative intensities of hybridization signals were obtained by densitometric scanning (RFLP-Scan software, Scanalytics, Inc.) of autoradiograms exposed within the linear range of the film (Eastman Kodak X-Omat). Human multiple tissue Northern blots were purchased from CLONTECH, hybridized, and washed according to manufacturer's instructions.

Proliferation Assay-- The protein coding region of the IRT-1 cDNA was cloned by PCR using IRT-1 cDNA sequence-specific primers. The PCR 5'-primer also contained a Kozak consensus sequence (GCCGCCGCCATGG) to enhance translation (27). This modified protein coding sequence was inserted into the expression vector pBK-CMV (Stratagene), and purified DNA from a single bacterial colony containing IRT-1 in pBK-CMV was isolated. This construct was termed pBK-CMV-IRT-1.

Human coronary artery smooth muscle cells grown in T75 flasks were transfected with no plasmid (mock control), with the pBK-CMV plasmid alone, or with pBK-CMV-IRT-1 in the forward and reverse orientations using 2 µl/ml LipofectAMINE reagent (Life Technologies, Inc.) and mixed with 1 µg of either plasmid. Two days following transfection, cells were trypsinized and split 1:2, with one-half grown in the presence of the neomycin analog G418 (Geneticin) and left to grow in the presence of growth medium + G418 for 14 days. The other half was saved for RNA isolation. After selection for 14 days, the cells were then trypsinized and counted using a standard hemocytometer.

Semiquantitative Reverse Transcription-Polymerase Chain Reaction-- Total RNA was extracted from transfected cells as described above, and 4 µg was reverse-transcribed using random hexamers as described previously (26). One-fifth of the cDNA was PCR-amplified for 32 cycles using the following neomycin-specific amplimers: 5'-GCAAGCAGGCATCGCCATGGTTCA-3' and 5'-TGGGCGAAGTGCCGGGGCAGGATC-3', which define a 290-base pair region of the neomycin cDNA. This is in the linear assay range with respect to cycle number, template concentration, and dilution of cDNA. The glyceraldehyde-3-phosphate dehydrogenase amplimers were purchased from CLONTECH and define an amplicon of 450 base pairs. One-fifth of the reaction was run on a 2.5% agarose gel, ethidium bromide-stained, and photographed.

Immunohistochemistry-- Stably transfected cells were grown on microscope slides. The medium was removed, and cells were rinsed with phosphate-buffered saline and fixed in 2% paraformaldehyde. Immunoperoxidase staining was performed using the Zymed Histostain-Plus kit. Cells were incubated in 0.1% hydrogen peroxide to quench endogenous peroxidase activity, in 10% nonimmune blocking serum for 15 min, and overnight at 4 °C in a 1:1000 dilution of column-purified IRT-1 primary antibody. Cells were washed, incubated with streptavidin-peroxidase enzyme conjugate, and incubated with aminoethyl carbazole chromogen. Cells were rinsed and counterstained with hematoxylin and mounted.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Expression of IRT-1 mRNA in VSMCs Is Interferon-gamma -specific-- IRT-1 was identified as an aberrant PCR product using gene-specific primers and RNA extracted from VSMCs stimulated with various cytokines as a template (26). Under low stringency primer annealing conditions, a PCR product almost twice the expected size of the gene we were studying was observed in samples from IFN-gamma -treated VSMCs (data not shown). Using this PCR product as a probe, we verified the expression pattern of its cognate cDNA by Northern analysis of VSMCs stimulated with various cytokines. Fig. 1 indicates that the transcript recognized by this probe is ~1300 nucleotides in length and, similar to that observed by reverse transcription-PCR, is expressed in these cells only upon treatment with IFN-gamma . Expression of this transcript in VSMCs is dependent upon IFN-gamma treatment, regardless of prior serum starvation (data not shown). This indicates that IRT-1 is an IFN-gamma -specific transcript.


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  		Fig. 1.   Expression of human IRT-1 mRNA in VSMCs is induced with IFN-gamma . VSMCs were serum-starved for 48 h; and 15 µg of total RNA was isolated from serum-starved VSMCs (lane 1) and from VSMCs treated for 24 h with 10% fetal calf serum (lane 2), basic fibroblast growth factor (lane 3), IFN-gamma (lane 4), IL1-beta (lane 5), platelet-derived growth factor (lane 6), and transforming growth factor-beta (lane 7). Total RNA was separated on a formaldehyde-containing 1.3% agarose gel, transferred to nitrocellulose, and hybridized with the selected probe. The same filter was stripped and sequentially hybridized with probes for the respective genes as shown, and the probes were exposed to film overnight.

IRT-1 Encodes a Protein Containing a Leucine Zipper and Nuclear Localization Sequence-- We determined the full-length IRT-1 transcript by the rapid amplification of cDNA ends procedure using IRT-1 sequence-specific primers. The full-length IRT-1 cDNA transcript is ~1.25 kilobase pairs (Fig. 2A) and, following termination codons in all three reading frames, contains on open reading frame of 399 nucleotides encoding for a deduced 132-amino acid basically charged protein (pI 9.89) with a mass of ~14,617 kDa. This open reading frame was confirmed by cell-free in vitro translation of both the full-length cDNA and the deduced open reading frame, each of which displayed the predicted 14-kDa protein (data not shown).


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  		Fig. 2.   A, cDNA and deduced amino acid sequences of human IRT-1. Potential phosphorylation consensus sequences for mitogen-activated protein kinase and protein kinase C at amino acids 67-70 and 81-83, respectively, are in boldface. The boxed area indicates the nuclear localization signal RPKK, and the leucine zipper motif (LX6LX6LX6LX6) is underlined. B, schematic representation of the IRT-1 protein. The nuclear localization sequence (RPKK) at amino acids 25-28 is denoted by an asterisk; the highly hydrophobic area from amino acids 50 to 70 is boxed and shaded; and the strongly basic area from amino acids 57 to 65 is filled. The leucine zipper motif (LX6LX6LX6LX6) from amino acids 75 to 95 is boxed and shaded. C, hydrophobicity profile of the deduced IRT-1 amino acid sequence according to the method of Kyte and Doolittle (23).

The deduced amino acid sequence of human IRT-1 contains a number of motifs that may suggest its function and is depicted schematically in Fig. 2B. A strongly basic region at amino acids 67-75 is immediately followed by a consensus leucine zipper motif (LX6LX6LX6LX6) at amino acids 75-95. This bZIP pattern is present in many gene regulatory proteins (28). A single strongly hydrophobic region is indicated in amino acids 50-80, and Fig. 2C is a Kyte-Doolittle depiction of this hydrophobicity. A 4-amino acid nuclear localization sequence (RPKK), identical to the SV40 large T antigen core sequence, is also present at amino acids 25-28 (29, 30). The amino acid sequence of this protein predicts a strong alpha -helix secondary structure, also prevalent in some gene regulatory proteins. Other amino acid domains include potential phosphorylation consensus sequences for mitogen-activated protein kinase and protein kinase C at amino acids 67-70 and 81-83, respectively. The IRT-1 cDNA also has a long 3'-untranslated region that contains an ATTTA sequence, which is found in the mRNA of many cytokines and proto-oncogenes and is thought to confer instability to mRNA (31, 32).

IRT-1 mRNA Expression Is Temporal and Cycloheximide-sensitive-- IRT-1 mRNA expression is dose-dependent, with optimal concentrations of IFN-gamma being 100 units/ml (data not shown). The time course of IRT-1 expression was also investigated. IRT-1 expression is temporal, beginning at ~8 h after IFN-gamma treatment and reaching a peak at 72 h after IFN-gamma treatment (Fig. 3). Quantitation of this expression by scanning densitometric analysis normalized to RNA content reveals a >200-fold induction of IRT-1 mRNA 72 h after IFN-gamma treatment (not shown).


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  		Fig. 3.   Time course of IRT-1 mRNA expression in IFN-gamma -stimulated human VSMCs. VSMCs were serum-starved for 48 h. 15 µg of total RNA isolated from unstimulated VSMCs (lane 1) and from VSMCs stimulated with 100 units/ml IFN-gamma for 4 h (lane 2), 8 h (lane 3), 16 h (lane 4), 24 h (lane 5), 48 h (lane 6), and 72 h (lane 7) was separated on a formaldehyde-containing 1.3% agarose gel; transferred to nitrocellulose; and hybridized with the IRT-1 cDNA probe.

Generally, IFN-gamma -inducible proteins are regulated at the transcriptional level in a protein synthesis-dependent fashion (33). To determine if IRT-1 transcription is dependent on protein synthesis, a 500 nM concentration of the protein synthesis inhibitor cycloheximide was added to VSMCs and then stimulated with IFN-gamma for 24 h. Fig. 4 shows that this concentration of cycloheximide inhibits expression of IRT-1 by ~96%, suggesting that transcription of IRT-1 mRNA is dependent on de novo synthesized transcription factors. The addition of cycloheximide to unstimulated VSMCs did not induce IRT-1 expression, suggesting that inhibition of transcription of this gene in unstimulated cells is not under the control of constitutively expressed factors.


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  		Fig. 4.   A, effect of cycloheximide on accumulation of IRT-1 mRNA. Human VSMCs were serum-starved for 48 h and then stimulated with IFN-gamma in the presence or absence of 500 ng/ml cycloheximide (CHX) for 24 h, and RNA was analyzed by Northern analysis. First lane, no IFN-gamma , no cycloheximide; second lane, no IFN-gamma , with cycloheximide; third lane, with IFN-gamma , no cycloheximide; fourth lane, with IFN-gamma , with cycloheximide. The blot was exposed to film overnight.

IRT-1 mRNA Is Expressed in Injured Rat Carotid Arteries-- Activated T lymphocytes, which are present in injured arteries, produce IFN-gamma , and a number of studies have determined that T lymphocytes exert pleiotropic effects on VSMCs (12, 13). Because activated VSMCs are the primary cell type in restenotic lesions, we hypothesized that this transcript would be prevalent in injured arteries. Total RNA from undamaged arteries, and from rat carotid arteries at three time points post-balloon angioplasty, was isolated and Northern analysis performed with IRT-1 cDNA as a hybridization probe. Fig. 5 demonstrates that IRT-1 mRNA is induced by balloon angioplasty in an acute fashion, with a 10-fold increase in expression of IRT-1 mRNA over basal levels 1 day post-balloon angioplasty, 3-fold at 3 days, and 2-fold at 7 days post-injury. This indicates that expression of this gene is induced in rat carotid arteries in response to balloon angioplasty.


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  		Fig. 5.   IRT-1 mRNA is induced by balloon angioplasty injury in rat carotid arteries. Shown are the results from Northern analysis of RNA from rat left common carotid arteries subjected to balloon angioplasty prior to and 1, 3, and 7 days following balloon angioplasty (lanes 1-4, respectively) and probed with a human IRT-1 DNA probe. Total RNA (10 µg) from rat carotid arteries was separated on a formaldehyde-containing 1.3% agarose gel, transferred to nitrocellulose, hybridized, and washed as described under "Materials and Methods."

IRT-1 mRNA Is Constitutively Expressed in Several Tissues-- To determine the tissue distribution of this transcript, filters containing RNA from 16 different human tissues were screened with the IRT-1 cDNA as a probe. IRT-1 mRNA is expressed in a variety of human tissues, with the highest expression in cells of lymphoid origin, in particular, spleen, pheripheral blood (PBLs), and thymus (Fig. 6). Other tissues expressing appreciable amounts of IRT-1 are lung, skeletal muscle, and small intestine. Varying but detectable amounts of expression are in pancreas, kidney, liver, placenta, heart, colon, ovary, testes, and prostate. No IRT-1 mRNA is detectable in brain. This pattern indicates that IRT-1 expression is tissue-specific, and the relatively high degree of constitutive expression of IRT-1 in human lymphoid tissue suggests a function for this protein in cells of this lineage.


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  		Fig. 6.   Northern analysis of human tissue distribution of human IRT-1 mRNA expression. Two micrograms of poly(A)+ mRNAs from pancreas (lane 1), kidney (lane 2), skeletal muscle (lane 3), liver (lane 4), lung (lane 5), placenta (lane 6), brain (lane 7), heart (lane 8), peripheral blood lymphocytes (lane 9), colon (lane 10), small intestine (lane 11), ovary (lane 12), testes (lane 13), prostrate (lane 14), thymus (lane 15), and spleen (lane 16) was hybridized with each respective probe as described under "Materials and Methods." The respective size of the transcript is 1.35 kilobase pairs, and size standards in kilobase pairs are indicated by the numbers on the right. The blot was purchased from CLONTECH and exposed to film overnight.

Constitutive IRT-1 Expression Is Reduced in Proliferating Lymphocytes-- The high degree of constitutive expression of IRT-1 in human lymphoid tissue led us to investigate if IRT-1 expression is regulated in activated human lymphocytes. Northern analysis of the IRT-1 transcript in unstimulated and PHA-stimulated human PBLs showed that unstimulated PBLs demonstrated a high level of constitutive IRT-1 expression, consistent with that observed in the multiple tissue analysis (Fig. 7A). However, a 24-h treatment of these cells with PHA decreased IRT-1 mRNA levels >3-fold, and by 72 h, IRT-1 mRNA levels were decreased 6-fold (Fig. 7B). As expected, proliferating cell nuclear antigen levels in such treated cells were increased dramatically, reflecting the proliferative state of these cells. These results indicate that the constitutive levels of IRT-1 mRNA expression in quiescent human PBLs can be diminished by the proliferative lymphocyte mitogen PHA.


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  		Fig. 7.   Expression of IRT-1 mRNA is down-regulated in PHA-activated human lymphocytes. A, PBLs isolated from a normal human donor were untreated (lane 1) or stimulated with 5 µg/ml PHA for 24 and 72 h (lanes 2 and 3, respectively). Ten micrograms of total RNA was separated on a formaldehyde-containing 1.3% agarose gel, transferred to nitrocellulose, and hybridized with the selected probe. The same filter was stripped and sequentially hybridized with probes for the respective mRNAs as shown. PCNA, proliferating cell nuclear antigen. B, densitometric analysis of IRT-1 expression in mitogen-stimulated human PBLs. Values expressed are displayed as the relative level of expression normalized to 28 S RNA.

Overexpression of IRT-1 Results in Inhibition of VSMC Growth and Altered Cell Morphology-- As an initial approach toward understanding the function of IRT-1, we forced expression of this gene in human VSMCs. The protein coding region of the IRT-1 cDNA was cloned by PCR using IRT-1 cDNA sequence-specific primers and a PCR 5'-primer containing the Kozak consensus sequence (GCCGCCGCCATGG) to enhance translation (27). This sequence increases IRT-1 protein expression 9-fold in an in vitro translation system (data not shown). Human vascular smooth muscle cells were transfected with no plasmid (mock control), with the pBK-CMV plasmid alone, or with pBK-CMV containing IRT-1 (pBK-CMV-IRT-1) in the forward and reverse orientations. Two days following transfection, cells were trypsinized and split 1:2, with one-half grown in the presence of the neomycin analog G418 (Geneticin) and left to grow in the presence of growth medium + G418 for 14 days. The other half was saved for RNA isolation. After selection for 14 days, the cells were then trypsinized and counted using a standard hemocytometer. The results of three experiments are tabulated in Table I and demonstrate an average 18% decrease in pBK-CMV-IRT-1-containing cells as compared with pBK-CMV and pBK-CMV-IRT-1 antisense orientation control cells. These results are not due to differences in transfection efficiency, as reverse transcription-PCR of RNA isolated from newly transfected cells indicated that equal amounts of the plasmid pBK-CMV were present in both plasmid-only and pBK-CMV-IRT-1-transfected cells (Fig. 8). Overexpression of other genes that do not significantly affect VSMC proliferation has no effect on cell number in this system (data not shown). This demonstrates that human VSMCs that overexpress IRT-1 proliferate at a dramatically slower rate than do cells that do not express IRT-1 protein, suggesting an antiproliferative function for IRT-1.

                              
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Table I
Overexpression of IRT-1 protein in human VSMCs inhibits their proliferation
Human coronary artery VSMCs were transfected with no plasmid (mock control), the pBK-CMV plasmid alone, pBK-CMV-IRT-1 in the antisense orientation, or pBK-CMV-IRT-1. Two days following transfection, cells were trypsinized, split 1:2, and left to grow in the presence of growth medium + G418 for 14 days. After selection for 14 days, the cells were trypsinized and counted using a standard hemocytometer. -Fold reduction refers to the cell number in vector-alone control/cell number in IRT-1-containing cells. The data are from three separate experiments.


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  		Fig. 8.   Reduction of proliferative response of VSMCs overexpressing IRT-1 is not due to differences in transfection efficiency. Total RNA was extracted from transfected cells 2 days after transfection, and 4 µg was reverse-transcribed and PCR-amplified using neomycin (Neo)-specific amplimers, which define a 290-base pair region of the neomycin cDNA, and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) amplimers, which define an amplicon of 450 base pairs. One-fifth of the reaction was electrophoresed on a 2.0% agarose gel, ethidium bromide-stained, and photographed. Lane 1, mock transfection; lane 2, pBK-CMV; lane 3, pBK-CMV-IRT-1; lane 4, pBK-CMV-IRT-1 (antisense); lane 5, neomycin control; lane 6, glyceraldehyde-3-phosphate dehydrogenase control; lane 7, no-reverse transcriptase control from IRT-1-transfected cells.

It was possible that viable VSMCs in the IRT-1-transfected samples were the result of the absence of or a reduced expression of IRT-1 in these cells. To determine if IRT-1 was being expressed in this population, human VSMCs from the above-described experiments were grown on slides, and immunohistochemistry was performed with antisera directed to amino acids 58-72 (KWERRERVSPPSPHP) present in the IRT-1 protein. Cells transfected with pBK-CMV alone displayed no staining, whereas every cell in the pBK-CMV-IRT-1 group clearly indicated a distinct perinuclear staining (Fig. 9). This experiment indicates that IRT-1 protein was indeed being expressed in VSMCs displaying growth inhibition and also identifies the cellular localization of transfected IRT-1 protein as perinuclear.


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  		Fig. 9.   Immunohistochemical localization of IRT-1 in transfected human VSMCs. A, pBK-CMV control cells; B, pBK-CMV-IRT-1-transfected cells. Cells transfected as described in the legend to Table I were trypsinized, grown on microscope slides for 24 h, and then subjected to immunohistochemistry with IRT-1-specific antisera as described under "Materials and Methods." Cells were rinsed and counterstained with hematoxylin and mounted. Magnification × 500.

In addition to reduced proliferative capability, VSMCs that overexpressed IRT-1 also displayed significant morphological differences from cells transfected with pBK-CMV alone (Fig. 9). VSMCs stably transfected with IRT-1 displayed a flattened, scallop-shape morphology, as opposed to the typical hill-and-valley morphology representative of normally growing VSMCs.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In this study, we describe IRT-1, a novel transcript that may represent an IFN-gamma -inducible gene regulatory factor in human VSMCs. The IRT-1 transcript is synthesized in human VSMCs only when they are IFN-gamma -treated (Fig. 1). Further analysis showed that IFN-gamma induces IRT-1 mRNA expression beginning at 8 h and maximizing at >200-fold 72 h after treatment (Fig. 3). This expression pattern is generally slower than that observed for other IFN-gamma -inducible proteins, such as IRF-1 (34), p48 (35), and IFP35 (36), and the Ia antigens (37), which peak 12, 18, and 24 h after IFN-gamma addition. The observation of peak IRT-1 levels at 72 h after stimulation suggests a function for this transcript in later events associated with IFN-gamma -induced gene expression.

Neointima formation subsequent to balloon angioplasty is the result of a dynamic process actively involving several different cell types and occurring in several phases. The initial response is primarily inflammatory in nature, involving T lymphocytes and macrophages, which secrete many different cytokines, including IFN-gamma , which are seminal to the local inflammatory response. Several studies have shown that in rats, IFN-gamma treatment inhibits arterial restenosis due to balloon angioplasty (24, 25). In these studies, intraperitoneal IFN-gamma treatment of rats for 7 days post-balloon angioplasty resulted in a 75% reduction of intimal VSMC proliferation and a 50% decrease in intima formation. This protection lasted to 10 weeks post-procedure, suggesting that IFN-gamma -mediated gene expression during the first week after vessel damage is crucial for vascular protection. In balloon-damaged rat carotid arteries, we observed a marked induction of IRT-1 expression 1 day post-angioplasty, which declined to half-maximal levels by 1 week (Fig. 5). This suggests that IRT-1 activity may play a role in mediating the initial stages of the restenotic lesion.

Similar to IFN-gamma -inducible proteins, IRT-1 expression requires protein synthesis and therefore is cycloheximide-sensitive. Low doses of cycloheximide (100 ng/ml) have been used to dissect growth factor-induced G1 progression (38) and, at this concentration, inhibit protein synthesis by 50% within 1 h and completely prevent cell cycle progression as well as expression of RNA coding thymidine kinase (39). In our hands, cycloheximide concentrations above 1 µg/ml show toxic effects on human VSMCs.2 In the presence of 500 ng/ml cycloheximide, acceptable cell viability is maintained, and IRT-1 expression is inhibited 94%, indicating the necessity of de novo synthesized IFN-gamma -responsive factors for transcription (Fig. 4). The observation that there is no expression in cycloheximide-treated, non-IFN-gamma -treated cells or a lack of superinduction in cycloheximide-treated and IFN-gamma -treated cells indicates that transcription of IRT-1 in untreated cells is not repressed by a factor dependent on protein synthesis. The IRT-1 3'-untranslated region does contain an ATTTA sequence, which is found in many cytokine and proto-oncogene mRNAs and is thought to confer instability to mRNA (31, 32); therefore, further modulation of IRT-1 mRNA levels by post-transcriptional mechanisms such as increased half-life cannot be ruled out.

IRT-1 maps to an intronic region of the human major histocompatibility complex class III gene, B cell activation transcript (BAT-2), located in the major histocompatibility complex region of chromosome 6 in humans (40). Since this region localizes to an intron of BAT-2, the IRT-1 mRNA is a novel transcript. The BAT-2 gene product itself is a large, proline-rich protein with no known function (41), and treatment of VSMCs with a number of cytokines, including IFN-gamma , does not alter its expression (data not shown). The major histocompatibility complex class III region is one of the more densely gene-packed regions of the human genome, an estimated 10 times more concentrated than other areas of the genome. The location and function of some of the genes clustered here (cell-surface glycoproteins, complement cascade proteins, heat shock proteins, tumor necrosis factor, and NF-kappa B) indicate that genes mapping to this region could contribute to immune function and disease pathophysiology. Therefore, the chromosomal location of IRT-1 indicates that it may play a role in inflammatory processes or immune regulation.

The open reading frame of the IRT-1 mRNA was confirmed by in vitro translation and displays the predicted 14-kDa protein. This protein contains a leucine zipper motif, in which the leucine side chains from each alpha -helix interact with those from a similar alpha -helix from a second polypeptide, facilitating dimerization (28, 42). The IRT-1 protein also contains a single strongly hydrophobic region adjacent to the leucine zipper that contains a strongly basic center, which may represent a protein- or DNA-binding site. In bZIP proteins, this configuration mediates protein dimerization through these domains with other alpha -helix-containing proteins to create the potential for a large repertoire of DNA-binding functional complexes (28). This bZIP pattern is present in many gene regulatory proteins, including ATF/cAMP response element-binding proteins (CREB), the Jun/AP1 transcription factor family, and Oct-2 octomer-binding transcription factor (28). Examples of IFN-gamma -inducible leucine zipper proteins are more limited. One such protein is IFP35, an IFN-gamma -inducible bZIP protein that has been shown to interact with B-ATF, a member of the AP1 class of transcription factors (36, 43). No function for this protein has been described. The IRT-1 protein contains a 4-amino acid nuclear localization sequence at amino acids 25-28 which is similar to the SV40 large T antigen core sequence; RPKK (29, 30). This domain may also implicate it as a gene regulatory protein.

The human tissue distribution of IRT-1 suggests a role in the functioning of several tissue types, particularly lymphoid tissue (Fig. 6). Indeed, the constitutive levels of IRT-1 in normally quiescent PBLs, when stimulated to proliferate, decreased 6-fold (Fig. 7). This suggests at least dual roles for this protein: 1) a constitutive function in the maintenance of the quiescent phenotype in nonproliferating lymphocytes and 2) an inducible proactive function in IFN-gamma -driven antiproliferative activity. This is also similar to that attributed to the growth-restraining function of transcription factor IRF-1. In NIH 3T3 cells, IRF-1 mRNA is constitutively expressed; however, after serum stimulation, IRF-1 mRNA is reduced 6-fold (44). The observed tissue-restricted expression, taken together with IFN-gamma -specific inducibility and suppression in proliferating lymphocytes, indicates the presence of complex cis-sequences and transactivators that tightly regulate IRT-1 transcription.

Because IFN-gamma inhibits proliferation of VSMCs in culture, we hypothesized that forced expression of IRT-1 in these cells would impart a growth modulatory effect as well. Indeed, stable transfectants containing IRT-1 protein proliferate at a dramatically slower rate than do cells that do not (Table I). These results are not due to differences in transfection efficiency, as reverse transcription-PCR of RNA isolated from freshly transfected cells indicated that equal amounts of the plasmid pBK-CMV were present in vector-only and pBK-CMV-IRT-1-transfected cells (Fig. 8).

There are several IFN-gamma -inducible genes that regulate cell growth, including IRF-1, IRF-2, and STAT1 (45, 46). Proteins of the IRF family interact with each other and with other families of transcription factors (15), which control the subsequent activation of IFN-regulated genes by interaction with specific cis-acting DNA elements. Deregulation of the relative ratios of IRF-1 and IRF-2 through modified expression leads to perturbation of cell proliferation and subsequent growth inhibition (44). In concordance, in NIH 3T3 cells, constitutive IRF-1 levels reduce after serum-stimulated proliferation, which is quite similar to the effect of PHA on IRT-1 levels in human PBLs (Fig. 6).

VSMCs that overexpress IRT-1 displayed a flattened, scallop-shape morphology, as opposed to the typical hill-and-valley morphology representative of normally growing VSMCs (Fig. 9). Morphological differences in cells overexpressing a growth-suppressive protein have been observed in other systems (47). Because of the limited number of cells available for study in these assays, it was necessary to determine that these cells expressed IRT-1 protein by immunostaining individual cells. Every cell in the stably transfected pBK-CMV-IRT-1 group demonstrated positive staining. The basic region and nuclear localization of IRT-1 would suggest it to be a nuclear protein. However, the perinuclear (rather than nuclear) immunolocalization of transfected IRT-1 in these cells does not immediately indicate a gene regulatory function, implying that this protein may not directly interact with DNA or may require IFN-gamma to induce its translocation into the nucleus.

IRT-1 is a novel transcript that may represent an IFN-gamma -inducible gene regulatory factor in human VSMCs. Thus far, three lines of experimental data suggest that IRT-1 may be involved in negative regulation of cell growth. 1) IRT-1 is expressed in VSMCs only when they are stimulated with the anti proliferative cytokine IFN-gamma . 2) IRT-1 is constitutively expressed in normally quiescent cells; however, when these cells are stimulated to proliferate, IRT-1 expression is substantially reduced. 3) The proliferative capability of VSMCs that overexpress IRT-1 is dramatically reduced. The bZIP configuration adjacent to a basic rich region along with a core nuclear localization sequence make it possible that this protein acts directly on, or in concert with, gene regulatory proteins that modulate cell growth.

    ACKNOWLEDGEMENTS

We thank Christopher Carbone, Neile Hartmann, and Kai Fu for technical assistance.

    FOOTNOTES

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

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U95213.

Dagger To whom correspondence should be addressed: Dept. of Cardiology and Physiology, Temple University School of Medicine, 3420 N. Broad St., Philadelphia, PA 19140.

1 The abbreviations used are: IFN, interferon; VSMC, vascular smooth muscle cell; IRF, interferon regulatory factor; PCR, polymerase chain reaction; bZIP, basic leucine zipper; PBL, peripheral blood lymphocyte; PHA, phytohemagglutinin A.

2 M. V. Autieri, unpublished observations.

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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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