From the Department of Molecular Biology, Deborah Heart and Lung Research Institute, Browns Mills, New Jersey 08015
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ABSTRACT |
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Interferon- (IFN-
) inhibits proliferation
of vascular smooth muscle cells (VSMCs) in culture and reduces arterial
restenosis post-balloon angioplasty. The identification and
characterization of IFN-
-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-
. This mRNA expression is induced >200-fold 72 h
after IFN-
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-
-inducible factor
that may regulate the progression of vascular proliferative
diseases.
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INTRODUCTION |
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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- (11, 12). The antiproliferative effects of
IFN-
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-
-inducible genes (14-16). IFN-
is also directly antiproliferative to VSMCs in tissue culture, and the
addition of IFN-
to proliferating VSMCs results in a reduction of
c-myc expression within 2 h (17). Other data suggest
that IFN-
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-
treatment. Finally, VSMCs co-cultured with endothelial cells transduced
with IFN-
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- 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-
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-
may represent
an antirestenotic cytokine therapy. Because interferon-
inhibits
proliferation of VSMCs in culture and IFN-
inhibits arterial
restenosis post-balloon angioplasty, identification and
characterization of IFN-
-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- 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-
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.
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MATERIALS AND METHODS |
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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-, 20 ng/ml
interleukin-1
, 20 ng/ml platelet-derived growth factor, or 2 ng/ml
transforming growth factor-
, 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-
, and transforming growth factor-
were purchased from
Life Technologies, Inc.; interleukin-1
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--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 -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
-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.
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RESULTS |
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Expression of IRT-1 mRNA in VSMCs Is
Interferon--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-
-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-
. Expression of this
transcript in VSMCs is dependent upon IFN-
treatment, regardless of
prior serum starvation (data not shown). This indicates that IRT-1 is
an IFN-
-specific transcript.
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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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IRT-1 mRNA Expression Is Temporal and
Cycloheximide-sensitive--
IRT-1 mRNA expression is
dose-dependent, with optimal concentrations of IFN-
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-
treatment and reaching a peak at
72 h after IFN-
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-
treatment (not shown).
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IRT-1 mRNA Is Expressed in Injured Rat Carotid
Arteries--
Activated T lymphocytes, which are present in injured
arteries, produce IFN-, 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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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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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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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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DISCUSSION |
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In this study, we describe IRT-1, a novel transcript that may
represent an IFN--inducible gene regulatory factor in human VSMCs.
The IRT-1 transcript is synthesized in human VSMCs only when they are
IFN-
-treated (Fig. 1). Further analysis showed that IFN-
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-
-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-
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-
-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-, which are seminal
to the local inflammatory response. Several studies have shown that in
rats, IFN-
treatment inhibits arterial restenosis due to balloon
angioplasty (24, 25). In these studies, intraperitoneal IFN-
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-
-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--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-
-responsive factors for
transcription (Fig. 4). The observation that there is no expression in
cycloheximide-treated, non-IFN-
-treated cells or a lack of superinduction in cycloheximide-treated and IFN-
-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-, 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-
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 -helix interact with those from a similar
-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
-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-
-inducible leucine zipper proteins are more limited.
One such protein is IFP35, an IFN-
-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--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-
-specific inducibility and suppression in proliferating
lymphocytes, indicates the presence of complex cis-sequences
and transactivators that tightly regulate IRT-1 transcription.
Because IFN- 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--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- to induce its translocation
into the nucleus.
IRT-1 is a novel transcript that may represent an IFN--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-
. 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.
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ACKNOWLEDGEMENTS |
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We thank Christopher Carbone, Neile Hartmann, and Kai Fu for technical assistance.
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FOOTNOTES |
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* 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.
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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REFERENCES |
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