FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2106031353> ?p ?o ?g. }

• W2106031353 endingPage "14737" @default.
• W2106031353 startingPage "14731" @default.
• W2106031353 abstract "Interferon-γ (IFN-γ) inhibits proliferation of vascular smooth muscle cells (VSMCs) in culture and reduces arterial restenosis post-balloon angioplasty. The identification and characterization of IFN-γ-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-γ. This mRNA expression is induced >200-fold 72 h after IFN-γ 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-γ-inducible factor that may regulate the progression of vascular proliferative diseases. Interferon-γ (IFN-γ) inhibits proliferation of vascular smooth muscle cells (VSMCs) in culture and reduces arterial restenosis post-balloon angioplasty. The identification and characterization of IFN-γ-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-γ. This mRNA expression is induced >200-fold 72 h after IFN-γ 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-γ-inducible factor that may regulate the progression of vascular proliferative diseases. 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 (1Libby P. Schwartz D. Brogi E. Tanaka H. Clinton S.K. Circulation. 1992; 86: 47-52Crossref PubMed Scopus (59) Google Scholar). 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 (2Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9947) Google Scholar). These cytokines include, but are not limited to, interleukin-1, platelet-derived growth factor, and a number of colony-stimulating factors and interferons (IFNs) 1The 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. 1The 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. (3O'Brien E.R. Alpers C.E. Stewart D.K. Ferguson M. Tran N. Gordon D. Benditt E.P. Hinohara T. Simpson J.B. Schwartz S.M. Circ. Res. 1993; 73: 223-231Crossref PubMed Scopus (330) Google Scholar, 4Clowes A.W. Reidy M.A. Clowes M.M. Lab. Invest. 1983; 49: 208-215PubMed Google Scholar). 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 (5Liu M.W. Roubin G.S. King S.B. Circulation. 1989; 79: 1374-1380Crossref PubMed Google Scholar, 6Austin G.E. Ratliff N.B. Hollman J. Tabei S. Phillips D.F. J. Am. Coll. Cardiol. 1985; 6: 369-375Crossref PubMed Scopus (485) Google Scholar). 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 (5Liu M.W. Roubin G.S. King S.B. Circulation. 1989; 79: 1374-1380Crossref PubMed Google Scholar, 6Austin G.E. Ratliff N.B. Hollman J. Tabei S. Phillips D.F. J. Am. Coll. Cardiol. 1985; 6: 369-375Crossref PubMed Scopus (485) Google Scholar, 7Schwartz R.S. Murphy J.G. Edwards W.D. Camrud A.R. Vlietstra R.E. Holmes D.R. Circulation. 1990; 82: 2190-2200Crossref PubMed Scopus (397) Google Scholar, 8Nilsson J. Cardiovasc. Res. 1992; 27: 1184-1189Crossref Scopus (120) Google Scholar). Upon interaction with its target cell, interferons induce expression of a number of IFN-specific genes (9Tanaka N. Taniguchi T. Adv. Immunol. 1992; 52: 263-281Crossref PubMed Scopus (73) Google Scholar), which manifest their biological activities by antiviral, immune modulatory, and antiproliferative effects (10Demayer E. Demayer-Guignard J. Interferons and Other Regulatory Cytokines. John Wiley & Sons, Inc., New York1988: 91-113Google Scholar). This is particularly true in VSMCs, as it has been shown that proliferation of these cells is inhibited by lymphocyte-specific factors, primarily IFN-γ (11Hansson G.K. Holm J. Holm S. Fotev Z. Hedrich H.J. Fingerle J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10534-19530Crossref Scopus (135) Google Scholar, 12Rolfe B.E. Campbell J.H. Smith N.J. Cheong M.W. Campbell G.R. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1204-1210Crossref PubMed Scopus (29) Google Scholar). The antiproliferative effects of IFN-γ on VSMCs can be exerted indirectly, though generation of nitric oxide (13Nunokawa Y. Tanaka S. Biochem. Biophys. Res. Commun. 1992; 188: 409-415Crossref PubMed Scopus (108) Google Scholar), or directly, though generation of the interferon regulatory factor (IRF) family of transcriptional regulators, which can act as activators or repressors of IFN-γ-inducible genes (14Tanaka N. Kawakami T. Taniguchi T. Mol. Cell. Biol. 1993; 13: 4531-4538Crossref PubMed Scopus (383) Google Scholar, 15Wang I.-M. Blanco J.C. Tsai S.Y. Tsai M.-J. Ozato K. Mol. Cell. Biol. 1996; 16: 6313-6324Crossref PubMed Scopus (61) Google Scholar, 16Kirchhoff S. Schaper F. Hauser H. Nucleic Acids Res. 1993; 21: 2881-2889Crossref PubMed Scopus (126) Google Scholar). IFN-γ is also directly antiproliferative to VSMCs in tissue culture, and the addition of IFN-γ to proliferating VSMCs results in a reduction of c-myc expression within 2 h (17Bennett M.R. Evan G.I. Newby A.C. Circ. Res. 1994; 74: 525-536Crossref PubMed Scopus (185) Google Scholar). Other data suggest that IFN-γ inhibits VSMC proliferation by blocking the transition from G0 to G1 (18Hansson G.K. Jonasson L. Holm J. Clowes M.M. Clowes A.W. Circ. Res. 1988; 63: 712-719Crossref PubMed Scopus (219) Google Scholar). 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 (19Yamada H. Ochi K. Nakada S. Nemoto T. Horiguchi-Yamada J. Mol. Cell. Biochem. 1994; 136: 117-123Crossref PubMed Scopus (22) Google Scholar) by IFN-γ treatment. Finally, VSMCs co-cultured with endothelial cells transduced with IFN-γ cDNA grew 30–70% slower than control cells (20Stopeck A.T. Vahedian M. Williams S.K. Cell Transplant. 1997; 6: 1-8Crossref PubMed Scopus (14) Google Scholar). Immune cells are present in the atherosclerotic lesion and appear in greater numbers immediately following balloon angioplasty-induced vascular injury (21Hansson G.K. Jonasson L. Seifert P.S. Stemme S. Arteriosclerosis. 1989; 9: 567-578Crossref PubMed Google Scholar). IFN-γ 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 (12Rolfe B.E. Campbell J.H. Smith N.J. Cheong M.W. Campbell G.R. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1204-1210Crossref PubMed Scopus (29) Google Scholar, 22Wang W. Chen H.J. Giedd K.N. Schwartz A. Cannon P.J. Rabbani L.E. Circ. Res. 1995; 77: 1095-1106Crossref PubMed Scopus (20) Google Scholar). Furthermore, several studies have shown that in rats, IFN-γ treatment inhibits arterial restenosis due to balloon angioplasty (24Hansson G.K. Holm J. Circulation. 1991; 84: 1266-1271Crossref PubMed Scopus (110) Google Scholar,25Castronuovo J.J. Guss S.B. Mysh D. Sawhney A. Wolff M. Gown A.M. Cardiovasc. Surg. 1995; 3: 463-468Crossref PubMed Scopus (15) Google Scholar), which is likely due to its antiproliferative effects on VSMCs. These findings have raised the possibility that IFN-γ may represent an antirestenotic cytokine therapy. Because interferon-γ inhibits proliferation of VSMCs in culture and IFN-γ inhibits arterial restenosis post-balloon angioplasty, identification and characterization of IFN-γ-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-responsivetranscript-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 (26Autieri M.V. Biochem. Biophys. Res. Commun. 1996; 228: 29-37Crossref PubMed Scopus (90) Google Scholar). IRT-1 mRNA is expressed in VSMCs only by IFN-γ 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-γ 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. VSMCs were obtained as a cryopreserved secondary culture from Clonetics Corp. (San Diego, CA) and subcultured in growth medium as described previously (26Autieri M.V. Biochem. Biophys. Res. Commun. 1996; 228: 29-37Crossref PubMed Scopus (90) Google Scholar). 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-γ, 20 ng/ml interleukin-1β, 20 ng/ml platelet-derived growth factor, or 2 ng/ml transforming growth factor-β, 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-γ, and transforming growth factor-β were purchased from Life Technologies, Inc.; interleukin-1β 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. Total RNA was isolated from IFN-γ-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. The cDNA clone obtained above was dideoxynucleotide-sequenced on both strands in its entirety (Sequenase, U. S. Biochemical Corp.) as described previously (26Autieri M.V. Biochem. Biophys. Res. Commun. 1996; 228: 29-37Crossref PubMed Scopus (90) Google Scholar). DNA and protein sequences were analyzed using the MacVector software package (International Biotechnologies, Inc.). Searches for sequence similarity were performed using the GenBank™ nucleic acid data base and Prosite protein data base through the Genetics Computer Group FASTA, BLAST, PROSITE, and PSORT programs. 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 (26Autieri M.V. Biochem. Biophys. Res. Commun. 1996; 228: 29-37Crossref PubMed Scopus (90) Google Scholar). 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 tissuein 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. 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 (26Autieri M.V. Biochem. Biophys. Res. Commun. 1996; 228: 29-37Crossref PubMed Scopus (90) Google Scholar). 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 α-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 β-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 fromCLONTECH, hybridized, and washed according to manufacturer's instructions. 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 (27Kozak M. Nucleic Acids Res. 1987; 15: 8125-8148Crossref PubMed Scopus (4159) Google Scholar). 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. Total RNA was extracted from transfected cells as described above, and 4 μg was reverse-transcribed using random hexamers as described previously (26Autieri M.V. Biochem. Biophys. Res. Commun. 1996; 228: 29-37Crossref PubMed Scopus (90) Google Scholar). 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. 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. 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 (26Autieri M.V. Biochem. Biophys. Res. Commun. 1996; 228: 29-37Crossref PubMed Scopus (90) Google Scholar). 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-γ-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-γ. Expression of this transcript in VSMCs is dependent upon IFN-γ treatment, regardless of prior serum starvation (data not shown). This indicates that IRT-1 is an IFN-γ-specific transcript. 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.2 A ) 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). 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. 2 B . A strongly basic region at amino acids 67–75 is immediately followed by a consensus leucine zipper motif (LX 6LX 6LX 6LX 6) at amino acids 75–95. This bZIP pattern is present in many gene regulatory proteins (28Landschultz W.H. Johnson P.F. McKnight S.L. Science. 1988; 240: 1759-1764Crossref PubMed Scopus (2525) Google Scholar). A single strongly hydrophobic region is indicated in amino acids 50–80, and Fig. 2 C 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 (29Kalderon D. Roberts B.L. Richardson W.D. Smith A.E. Cell. 1984; 39: 499-509Abstract Full Text PDF PubMed Scopus (1858) Google Scholar, 30Kalderon D. Richardson W.D. Markham A.F. Smith A.E. Nature. 1984; 311: 33-38Crossref PubMed Scopus (907) Google Scholar). The amino acid sequence of this protein predicts a strong α-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 (31Caput D. Beutler B. Hartog K. Thayer R. Brown-Schimer S. Cerami A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1670-1674Crossref PubMed Scopus (1210) Google Scholar, 32Shaw G. Kamen R. Cell. 1986; 46: 659-667Abstract Full Text PDF PubMed Scopus (3112) Google Scholar). IRT-1 mRNA expression is dose-dependent, with optimal concentrations of IFN-γ 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-γ treatment and reaching a peak at 72 h after IFN-γ 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-γ treatment (not shown). Generally, IFN-γ-inducible proteins are regulated at the transcriptional level in a protein synthesis-dependent fashion (33Williams J.G. Jurkovich G.J. Maier R.V. J. Surg. Res. 1993; 54: 79-93Abstract Full Text PDF PubMed Scopus (73) Google Scholar). 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-γ 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. Activated T lymphocytes, which are present in injured arteries, produce IFN-γ, and a number of studies have determined that T lymphocytes exert pleiotropic effects on VSMCs (12Rolfe B.E. Campbell J.H. Smith N.J. Cheong M.W. Campbell G.R. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1204-1210Crossref PubMed Scopus (29) Google Scholar, 13Nunokawa Y. Tanaka S. Biochem. Biophys. Res. Commun. 1992; 188: 409-415Crossref PubMed Scopus (108) Google Scholar). 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. 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. 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. 7 A ). 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. 7 B ). 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. 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 (27Kozak M. Nucleic Acids Res. 1987; 15: 8125-8148Crossref PubMed Scopus (4159) Google Scholar). 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.Table IOverexpression of IRT-1 protein in human VSMCs inhibits their proliferationExp.MockpBK-CMVAntisense pBK-CMV-IRT-1pBK-CMV-IRT-1Reductioncells/flaskcells/flaskcells/flaskcells/flask-fold106.5 × 1057.1 × 1053.1 × 10420.0202.3 × 1061.9 × 1068.5 × 10527.5302.4 × 1052.2 × 1053.1 × 1047.7Human 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 IR" @default.
• W2106031353 created "2016-06-24" @default.
• W2106031353 creator A5005917520 @default.
• W2106031353 creator A5062970604 @default.
• W2106031353 date "1998-06-01" @default.
• W2106031353 modified "2023-09-30" @default.
• W2106031353 title "IRT-1, a Novel Interferon-γ-responsive Transcript Encoding a Growth-suppressing Basic Leucine Zipper Protein" @default.
• W2106031353 cites W1490497276 @default.
• W2106031353 cites W1516222795 @default.
• W2106031353 cites W1974456051 @default.
• W2106031353 cites W1975304761 @default.
• W2106031353 cites W1975511017 @default.
• W2106031353 cites W1976415203 @default.
• W2106031353 cites W1986420787 @default.
• W2106031353 cites W1989017709 @default.
• W2106031353 cites W1993625863 @default.
• W2106031353 cites W2005393972 @default.
• W2106031353 cites W2007244655 @default.
• W2106031353 cites W2008619553 @default.
• W2106031353 cites W2011476242 @default.
• W2106031353 cites W2017713266 @default.
• W2106031353 cites W2018898580 @default.
• W2106031353 cites W2024836429 @default.
• W2106031353 cites W2026581670 @default.
• W2106031353 cites W2029275361 @default.
• W2106031353 cites W2029535545 @default.
• W2106031353 cites W2033722975 @default.
• W2106031353 cites W2045348184 @default.
• W2106031353 cites W2051904824 @default.
• W2106031353 cites W2053796273 @default.
• W2106031353 cites W2057805062 @default.
• W2106031353 cites W2064670731 @default.
• W2106031353 cites W2080532845 @default.
• W2106031353 cites W2083935741 @default.
• W2106031353 cites W2084645021 @default.
• W2106031353 cites W2090162429 @default.
• W2106031353 cites W2091797313 @default.
• W2106031353 cites W2094415848 @default.
• W2106031353 cites W2094547999 @default.
• W2106031353 cites W2094743727 @default.
• W2106031353 cites W2136319177 @default.
• W2106031353 cites W2137570167 @default.
• W2106031353 cites W2144093304 @default.
• W2106031353 cites W2149640683 @default.
• W2106031353 cites W2163853656 @default.
• W2106031353 cites W2173560213 @default.
• W2106031353 cites W259726011 @default.
• W2106031353 cites W4211053163 @default.
• W2106031353 cites W4229547792 @default.
• W2106031353 doi "https://doi.org/10.1074/jbc.273.24.14731" @default.
• W2106031353 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9614071" @default.
• W2106031353 hasPublicationYear "1998" @default.
• W2106031353 type Work @default.
• W2106031353 sameAs 2106031353 @default.
• W2106031353 citedByCount "15" @default.
• W2106031353 countsByYear W21060313532012 @default.
• W2106031353 crossrefType "journal-article" @default.
• W2106031353 hasAuthorship W2106031353A5005917520 @default.
• W2106031353 hasAuthorship W2106031353A5062970604 @default.
• W2106031353 hasBestOaLocation W21060313531 @default.
• W2106031353 hasConcept C101762097 @default.
• W2106031353 hasConcept C104317684 @default.
• W2106031353 hasConcept C105782903 @default.
• W2106031353 hasConcept C11413529 @default.
• W2106031353 hasConcept C125411270 @default.
• W2106031353 hasConcept C150194340 @default.
• W2106031353 hasConcept C156860981 @default.
• W2106031353 hasConcept C157880135 @default.
• W2106031353 hasConcept C169760540 @default.
• W2106031353 hasConcept C185592680 @default.
• W2106031353 hasConcept C19408993 @default.
• W2106031353 hasConcept C202908374 @default.
• W2106031353 hasConcept C2776178377 @default.
• W2106031353 hasConcept C2776580952 @default.
• W2106031353 hasConcept C41008148 @default.
• W2106031353 hasConcept C515207424 @default.
• W2106031353 hasConcept C54355233 @default.
• W2106031353 hasConcept C70721500 @default.
• W2106031353 hasConcept C86339819 @default.
• W2106031353 hasConcept C86803240 @default.
• W2106031353 hasConcept C94966510 @default.
• W2106031353 hasConceptScore W2106031353C101762097 @default.
• W2106031353 hasConceptScore W2106031353C104317684 @default.
• W2106031353 hasConceptScore W2106031353C105782903 @default.
• W2106031353 hasConceptScore W2106031353C11413529 @default.
• W2106031353 hasConceptScore W2106031353C125411270 @default.
• W2106031353 hasConceptScore W2106031353C150194340 @default.
• W2106031353 hasConceptScore W2106031353C156860981 @default.
• W2106031353 hasConceptScore W2106031353C157880135 @default.
• W2106031353 hasConceptScore W2106031353C169760540 @default.
• W2106031353 hasConceptScore W2106031353C185592680 @default.
• W2106031353 hasConceptScore W2106031353C19408993 @default.
• W2106031353 hasConceptScore W2106031353C202908374 @default.
• W2106031353 hasConceptScore W2106031353C2776178377 @default.
• W2106031353 hasConceptScore W2106031353C2776580952 @default.
• W2106031353 hasConceptScore W2106031353C41008148 @default.
• W2106031353 hasConceptScore W2106031353C515207424 @default.
• W2106031353 hasConceptScore W2106031353C54355233 @default.

• next