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Interferon Regulatory Factor-1 Up-regulates Angiotensin II Type 2 Receptor and Induces Apoptosis

1997; Elsevier BV; Volume: 272; Issue: 18 Linguagem: Inglês

10.1074/jbc.272.18.11952

1083-351X

Masatsugu Horiuchi, Takehiko Yamada, Wataru Hayashida, Victor J. Dzau,

interferon and immune responses

The expression of the angiotensin II type 2 (AT2) receptor is developmentally and growth regulated. In cultured R3T3 cells, expression of this receptor is markedly induced at the confluent state and with serum deprivation. In this study we demonstrated that the removal of serum from culture media resulted in the induction of apoptosis in these cells and the addition of angiotensin II further enhanced apoptosis. We have previously identified an interferon regulatory factor (IRF) binding motif in the mouse AT2 receptor gene promoter region. In this report, we observed that serum removal increased IRF-1 expression, with a rapid and transient decrease of IRF-2. To prove that the changes in IRFs after serum removal mediated apoptosis and up-regulated AT2receptor, we transfected antisense oligonucleotides for IRF-1 or IRF-2 into R3T3 cells and observed that IRF-1 antisense oligonucleotide attenuated apoptosis and abolished the up-regulation of AT2receptor. IRF-2 antisense oligonucleotide pretreatment did not affect the onset of apoptosis after serum removal; instead, it increased AT2 receptor binding and enhanced angiotensin II-mediated apoptosis. Taken together, these results suggest that increased IRF-1 after serum starvation contributes to the induction of apoptosis and that increased IRF-1 up-regulates the AT2 receptor expression after serum starvation, resulting in enhanced angiotensin II-mediated apoptosis. The expression of the angiotensin II type 2 (AT2) receptor is developmentally and growth regulated. In cultured R3T3 cells, expression of this receptor is markedly induced at the confluent state and with serum deprivation. In this study we demonstrated that the removal of serum from culture media resulted in the induction of apoptosis in these cells and the addition of angiotensin II further enhanced apoptosis. We have previously identified an interferon regulatory factor (IRF) binding motif in the mouse AT2 receptor gene promoter region. In this report, we observed that serum removal increased IRF-1 expression, with a rapid and transient decrease of IRF-2. To prove that the changes in IRFs after serum removal mediated apoptosis and up-regulated AT2receptor, we transfected antisense oligonucleotides for IRF-1 or IRF-2 into R3T3 cells and observed that IRF-1 antisense oligonucleotide attenuated apoptosis and abolished the up-regulation of AT2receptor. IRF-2 antisense oligonucleotide pretreatment did not affect the onset of apoptosis after serum removal; instead, it increased AT2 receptor binding and enhanced angiotensin II-mediated apoptosis. Taken together, these results suggest that increased IRF-1 after serum starvation contributes to the induction of apoptosis and that increased IRF-1 up-regulates the AT2 receptor expression after serum starvation, resulting in enhanced angiotensin II-mediated apoptosis. Angiotensin II (Ang II) 1The abbreviations used are: Ang II, angiotensin II; AT1 receptor, angiotensin II type 1 receptor; AT2 receptor, angiotensin II type 2 receptor; IRF, interferon regulatory factor; PCR, polymerase chain reaction; kb, kilobase pair(s); iNOS, inducible nitric oxide synthase; EF, embryonic fibroblast; IRF-E, IRF-binding DNA element. exerts various actions in its diverse target tissues controlling vascular tone, hormone secretion, tissue growth, and neuronal activities. Most of the known effects of Ang II in adult tissues are mediated by the Ang II type 1 (AT1) receptor. Recently, a second receptor subtype known as AT2 receptor has been cloned by us and others (1Mukoyama M. Nakajima M. Horiuchi M. Sasamura H,. Pratt R.E. Dzau V.J. J. Biol. Chem. 1993; 268: 24539-24542Google Scholar, 2Kambayashi Y. Bardhan S. Takahashi K. Tsuzuki S. Inui H. Hamakubo T. Inagami T. J. Biol. Chem. 1993; 268: 24543-24546Google Scholar). The AT2 receptor is abundantly and widely expressed in fetal tissues, but present only in at limited levels in adult tissues such as adrenal medulla, specific brain regions, uterine myometrium, heart, and atretic ovarian follicles (3Daud A.I. Bumpus F.M. Husain A. Endocrinology. 1988; 122: 2727-2734Google Scholar, 4Grady E.F. Sechi L.A. Griffin C.A. Schambelan M. Kalinyak J.E. J. Clin. Invest. 1991; 88: 921-933Google Scholar, 5Tsutsumi K. Saavedra J.M. Am. J. Physiol. 1991; 261: H667-H670Google Scholar, 6Tsutsumi K. Stromberg C. Viswanathan M. Saavedra J.M. Endocrinology. 1991; 129: 1075-1082Google Scholar, 7Millan M.A. Jacobowitz D.M. Aguilera G. Catt K.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11440-11444Google Scholar, 8Pucell A.G. Hodges J.C. Sen I. Bumpus F.M. Husain A. Endocrinology. 1991; 128: 1947-1959Google Scholar). The highly abundant expression of this receptor during embryonic and neonatal growth and quick disappearance after birth has led to the suggestion that this receptor is involved in growth, development, and/or differentiation. Our studies using in vivo gene transfer of the rat AT2 receptor into an injured rat carotid artery have demonstrated an attenuation of neointimal hyperplasia resulting from AT2 receptor transgene overexpression (9Nakajima, M., Hutchinson, H., Fujinaga, M., Hayashida, W., Morishita, R., Zhang, L., Horiuchi, M., Pratt, R. E., and Dzau, V. J. (1997)Proc. Natl. Acad. Sci. U. S. A., in press.Google Scholar), and Stollet al. (10Stoll M. Steckelings M. Paul M. Bottari S.P. Metzger R. Unger T. J. Clin. Invest. 1995; 95: 651-65712Google Scholar) also demonstrated that the antiproliferative actions of the AT2 receptor offset the growth promoting effects mediated by the AT1 receptor. Greater than 99.9% of the ovarian follicles present at birth undergo atresia, an event dependent on apoptosis or "programmed cell death." Interestingly, AT2 receptor expression is tightly associated with ovarian follicular atresia (3Daud A.I. Bumpus F.M. Husain A. Endocrinology. 1988; 122: 2727-2734Google Scholar, 8Pucell A.G. Hodges J.C. Sen I. Bumpus F.M. Husain A. Endocrinology. 1991; 128: 1947-1959Google Scholar). AT2receptor is also abundantly expressed in immature brain and some specific regions of the brain (5Tsutsumi K. Saavedra J.M. Am. J. Physiol. 1991; 261: H667-H670Google Scholar, 6Tsutsumi K. Stromberg C. Viswanathan M. Saavedra J.M. Endocrinology. 1991; 129: 1075-1082Google Scholar). Approximately half of the neurons produced during embryogenesis die by apoptosis before adulthood. These results have led us to examine the role of AT2 receptor in apoptosis. We recently demonstrated that the activation of the AT2 receptor induces apoptosis in a rat pheochromocytoma cell line (PC12W) and confluent mouse fibroblast cell line (R3T3 cell) (11Yamada T. Horiuchi M. Dzau V.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 156-160Google Scholar). These cells have been shown to express abundant AT2receptor (12Speth R.C. Kim K.H. Biochem. Biophys. Res. Commun. 1990; 169: 997-1006Google Scholar, 13Dudley D.T. Hubbell S.E. Summerfelt R.M. Mol. Pharmacol. 1991; 40: 360-367Google Scholar, 14Dudley D.T. Summerfelt R.M. Regul. Pept. 1993; 44: 199-206Google Scholar). We further observed that higher AT2receptor expression is especially associated with apoptosis (11Yamada T. Horiuchi M. Dzau V.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 156-160Google Scholar). In R3T3 cells, AT2 receptor expression is regulated by the growth state of the cells. AT2 receptor binding density is very low in actively growing cells, but increases markedly in the confluent, quiescent state (13Dudley D.T. Hubbell S.E. Summerfelt R.M. Mol. Pharmacol. 1991; 40: 360-367Google Scholar, 14Dudley D.T. Summerfelt R.M. Regul. Pept. 1993; 44: 199-206Google Scholar). We recently reported that the growth-regulated AT2 receptor expression in R3T3 cells is transcriptionally controlled by the competitive binding of interferon regulatory factor-1 (IRF-1) and IRF-2 for an IRF binding sequence in the AT2 receptor promoter region (15Horiuchi M. Koike G. Yamada T. Mukoyama M. Nakajima M. Dzau V.J. J. Biol. Chem. 1995; 270: 20225-20230Google Scholar). IRF-1 and IRF-2 are structurally related, particularly in their amino-terminal regions that confer DNA binding specificity. They recognize the same DNA sequence elements (IRF-Es) (16Harada H. Fujita T. Miyamoto M. Kimura Y. Maruyama M. Furia A. Miyata T. Taniguchi T. Cell. 1989; 58: 729-739Google Scholar, 17Tanaka N. Kawakami T. Taniguchi T. Mol. Cell. Biol. 1993; 13: 4531-4538Google Scholar). IRF-1 functions as a transcriptional activator, whereas IRF-2 represses IRF-1 function by competing for the same cis-element (16Harada H. Fujita T. Miyamoto M. Kimura Y. Maruyama M. Furia A. Miyata T. Taniguchi T. Cell. 1989; 58: 729-739Google Scholar, 17Tanaka N. Kawakami T. Taniguchi T. Mol. Cell. Biol. 1993; 13: 4531-4538Google Scholar, 18Fujita T. Kinura Y. Miyamoto M. Barsoumian E.L. Taniguchi T. Nature. 1989; 337: 270-272Google Scholar, 19Harada H. Willison K. Sakakibara J. Miyamoto M. Fujita T. Taniguchi T. Cell. 1990; 63: 303-312Google Scholar). It has been shown that the growth state of certain cells depends on a balance between IRF-1 and IRF-2,i.e. a change in the IRF-1/IRF-2 ratio perturbs cell growth control (20Yamada G. Ogawa M. Akagi K. Miyamoto H. Nakano N. Itoh S. Miyazaki J. Nishikawa S. Yamamura K. Taniguchi T. Proc. Natl. Acad. Sci. U. S. A. 1990; 88: 532-536Google Scholar, 21Watanabe N. Sakakibara J. Hovanessian A.G. Taniguchi T. Fujita T. Nucleic Acids Res. 1991; 19: 4421-4428Google Scholar, 22Kirchhoff S. Schaper F. Hauser H. Nucleic Acids Res. 1993; 21: 2881-2889Google Scholar, 23Harada H. Kitagawa M. Tanaka N. Yamamoto H. Harada K. Ishihara M. Taniguchi T. Science. 1993; 259: 971-974Google Scholar). In this study, we examined the role of AT2 receptor expression in IRF-regulated apoptosis. We hypothesized that changes in the ratio of IRFs under specific conditions regulate AT2receptor expression which subsequently influences apoptosis. We studied the effect of serum starvation on R3T3 cells and observed that an increase in IRF-1 with transient decrease in IRF-2 stimulated AT2 receptor expression and enhanced AT2receptor-mediated apoptosis. R3T3 cells were cultured as described (13Dudley D.T. Hubbell S.E. Summerfelt R.M. Mol. Pharmacol. 1991; 40: 360-367Google Scholar) using Dulbecco's modified essential medium supplemented with 10% fetal bovine serum. Cells were seeded at 7 × 104cells/T-75 flask (Falcon) for transfection experiments for internucleosomal DNA fragmentation (DNA laddering), and cells were seeded on a 24-well plate (surface area, 2 cm2) (Falcon) at 2.5 × 104 cells/well for receptor binding studies. Two days after reaching confluent state, the cells were used for experiments. Cell number was counted by Coulter counter. Oligonucleotides used for treating cultured R3T3 cells as "decoy" include the following: IRF binding, 5′-GAAAAAGAGAAAGAGAAAATTCTGCTAAAAATGATA-3′ mutant IRF binding, 5′-GAAAAAAGGAAAAGGAAAATTCTGCTAAAAAGGATA-3′. In mutant IRF-binding oligonucleotide, three repeats of putative IRF binding hexamer motif as previously reported (15Horiuchi M. Koike G. Yamada T. Mukoyama M. Nakajima M. Dzau V.J. J. Biol. Chem. 1995; 270: 20225-20230Google Scholar) were replaced with C4 oligomer (AAAGGA), which does not bind to IRFs (16Harada H. Fujita T. Miyamoto M. Kimura Y. Maruyama M. Furia A. Miyata T. Taniguchi T. Cell. 1989; 58: 729-739Google Scholar). Oligonucleotides were annealed to complementary sequences and used as double-stranded oligonucleotides. Phosphorothioate oligonucleotides for treating cells as antisense DNA for IRF-1 and IRF-2 are as follows: IRF-1 antisense, 5′-GAAAGATGCCCGAGATGC-3′ (−111/−94) (24Miyamoto M. Fujita T. Kimura Y. Maruyama M Harada H. Sudo Y. Miyata T. Taniguchi T. Cell. 1988; 54: 903-913Google Scholar), IRF-2 antisense, 5′-GTGTGAGTGTTGTTAGGG-3′ (−71/−54) (16Harada H. Fujita T. Miyamoto M. Kimura Y. Maruyama M. Furia A. Miyata T. Taniguchi T. Cell. 1989; 58: 729-739Google Scholar). On the day of transfection, the medium was changed to fresh medium and incubated for 1 h at 37 °C. The transfection was performed using LipofectAMINE Reagent (Life Technologies, Inc.), 3:1 (w/w) liposome formulation of the polycationic lipid 2,3-dioleyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA) and the neutral lipid dioleoyl phosphatidylethanolamine (DOPE), according to the manufacturer's instructions. Cells were incubated with the oligonucleotides (0.5 μm) and LipofectAMINE (1:3, w/w) for 3 h, and the transfection reagent was replaced with culture medium as described in each figure legend. Cells on the wells were washed twice with 500 μl of phosphate-buffered saline containing 0.1% bovine serum albumin. Reactions were performed with 0.2 nm125I-CGP42112A (2,176 Ci mmol−1, Peptide Radioiodination Center, Washington State University) at 37 °C for 1 h. After incubation, cells were washed with ice-cold phosphate-buffered saline twice and solubilized with 200 μl of 0.5n NaOH and solubilized material was counted for radioactivity. Nonspecific binding was defined in the presence of 1 μm unlabeled CGP42112A. Total RNA was prepared from cultured R3T3 cells using RNAzol (Tel-Test), reverse transcribed using reverse transcriptase and random hexamers (Perkin-Elmer), and applied to PCR. PCR primers for IRF-1 used were as follows: 5′-CCTGATGACCACAGCAGTTAC-3′ and 5′-CTTCATCTCCGTGAAGACATG-3′. For IRF-2, PCR primers were 5′-GCGGTCCTGACTTCAGCTATA-3′ and 5′-CTTCTTGATGACACTGGCCGG-3′. PCR reaction was carried out with 30 cycles of 1 min f denaturation at 94 °C, 1 min of annealing at 55 °C, and 3 min of extension at 72 °C, followed by 15 min of final extension step. PCR-amplified DNAs were subcloned into pCR™II Vector (Invitrogen). cDNA probes for Northern blotting were prepared from these plasmid vector by restriction enzyme cut by EcoRI. Total RNA was prepared from cultured R3T3 cells using RNAzol (Tel-Test) 24 h after oligonucleotide treatment. RNA (20 μg/lane) was separated by electrophoresis and transferred onto a nylon membrane (Amersham), and hybridization was carried out using a 32P-labeled IRF-1 and IRF-2 cDNA, a32P-labeled HindIII-NsiI fragment of mouse AT2 receptor cDNA (25Nakajima M. Mukoyama M. Pratt R.E. Horiuchi M. Dzau V.J. Biochem. Biophys. Res. Commun. 1993; 197: 393-399Google Scholar), or 0.78-kbPstI-XbaI fragment of a human glyceraldehyde-3-phosphate dehydrogenase clone. Densitometric analysis of autoradiograms was performed by scanning densitometer (GS300, Hoeffer) and NIH Image software. R3T3 cells were seeded onto 10-cm dishes (Falcon) at 2 × 10−6 cells/dish for this experiment. The cells were washed with HEPES-buffered saline and lysed in 0.5 ml of phosphate-buffered saline containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mmphenylmethylsulfonyl fluoride, 1 mm sodium orthovanadate, and 10 μg/ml aprotinin. Cell lysates were clarified by centrifugation at 8,500 × g for 20 min, boiled in Laemmli loading buffer for 3 min, resolved by 12% SDS-polyacrylamide gel electrophoresis, electroblotted onto nitrocellulose membrane, and immunoblotted with IRF-1 or IRF-2 antibody (Santa Cruz Biotechnology). Antibodies were detected by horseradish peroxidase-linked secondary antibody using the ECL (enhanced chemiluminescence) system (Amersham). DNA extraction, subsequent 3′ end-labeling of DNA, gel electrophoresis, and quantitation of DNA fragmentation were performed as described previously (26Tilly J.L. Hsueh A.J.W. J. Cell Physiol. 1993; 154: 519-526Google Scholar). Briefly, 500 ng of DNA prepared from each treated cells was end-labeled with [α-32P]ddATP (Amersham) and terminal transferase (Boehringer Mannheim) for 60 min at 37 °C. Labeled DNA was loaded onto a 2% agarose gel, separated by electrophoresis, and autoradiographed. The amount of radiolabeled ddATP incorporated into low (<20 kb) molecular weight DNA fractions were quantitated by cutting the respective fraction of DNA from the dried gel and counting in a β-counter. The results were expressed as percentage of the counts in control samples as described in each figure legend. R3T3 cells seeded onto six-well plates (Falcon) were used for this experiment. Chromatin binding dyes (Hoechst 33342 and propidium iodide (Molecular Probes)) were added to the serum-free medium at a concentration of 5 or 10 μm to examine the morphological changes of nuclei. After incubation at 37 °C for 1 h, cells were collected. After centrifugation, the pellet was resuspended in phosphate-buffered saline and cells were viewed under UV microscopy. All values are expressed as mean ± S.D. Statistical significance was assessed by analysis of variance (ANOVA) followed by Scheffe's test. p < 0.01 was considered to be significant. We first examined the changes of IRF-1 and IRF-2 gene expressions after serum removal which induces apoptosis in this cell line (11Yamada T. Horiuchi M. Dzau V.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 156-160Google Scholar). As shown in Fig. 1, expression of IRF-1 increased in a time-dependent manner and showed a maximum at 24 h after serum removal, whereas IRF-2 mRNA transiently decreased 3 h after serum removal and returned to basal level at 24 h. To document that these change in IRF-1 and IRF-2 contributed to the increased AT2 receptor expression, we treated confluent R3T3 cells with double-stranded oligonucleotides spanning the IRF-Es in the mouse AT2receptor promoter region (15Horiuchi M. Koike G. Yamada T. Mukoyama M. Nakajima M. Dzau V.J. J. Biol. Chem. 1995; 270: 20225-20230Google Scholar) as transcriptional factor decoy, which could bound with IRF-1 and IRF-2 in R3T3 cells (15Horiuchi M. Koike G. Yamada T. Mukoyama M. Nakajima M. Dzau V.J. J. Biol. Chem. 1995; 270: 20225-20230Google Scholar). This strategy is based on the competition for binding to a specific transcription factor between a cis-element present in an endogenous target gene and an exogenously added oligonucleotide corresponding to thatcis-sequence. One day after transfection of the oligonucleotides, the cells were placed in serum-free medium, and AT2 receptor binding was determined 3 days later by radioligand receptor binding assay. As shown in Fig.2 A, serum depletion enhanced AT2receptor binding. Pretreatment with IRF-binding decoy oligonucleotide attenuated the increase of AT2 receptor in response to serum removal. IRF-binding decoy oligonucleotide treatment did not change the AT2 receptor density in serum-fed R3T3 cells. Mutant IRF-binding decoy oligonucleotide treatment did not affect AT2 receptor density. We also measured AT2receptor mRNA levels at 24 h after serum removal and observed that pretreatment with IRF-binding decoy oligonucleotide inhibited the AT2 receptor mRNA increase (Fig. 2 B), supporting the contention that the change in the ratio of IRF-1/IRF-2 after serum depletion is important for the transcriptional up-regulation of AT2 receptor expression.Figure 2Effects of oligonucleotides spanning IRF binding sequence in mouse AT2 receptor promoter region and antisense oligonucleotides for IRF-1 and IRF-2 on AT2receptor expression after serum starvation. Two double-stranded oligonucleotides (IRF and mutant IRF-binding decoy oligonucleotides) (see "Experimental Procedures") were transfected into confluent R3T3 cells, and 1 day after oligonucleotides transfection, serum was removed from the medium. The AT2 receptor binding was determined 3 days after serum removal by radioligand binding assay.A, the values were expressed as mean ± S.D. obtained from five different culture wells. Total RNA was prepared from R3T3 cells 24 h after serum removal or serum-fed cells. RNA (20 μg/lane) was separated by electrophoresis and hybridization was carried with a probe for the AT2 receptor or for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control. Similar results were obtained in three different experiments (B). Antisense oligonucleotides for IRF-1 and IRF-2 were transfected into R3T3 cells. One day after oligonucleotide transfection, serum was removed from the medium. The AT2 receptor binding was determined 3 days after serum removal by radioligand binding assay (C). The values were expressed as mean ± S.D. obtained from five different cell culture wells. * shows p < 0.01 compared with LipofectAMINE transfection alone.View Large Image Figure ViewerDownload (PPT) To prove that the changes of IRF-1 and IRF-2 expression after serum depletion regulates the AT2 receptor expression, we transfected antisense oligonucleotides for IRF-1 or IRF-2 into R3T3 cells 24 h before serum removal. To confirm whether these antisense oligonucleotides inhibit IRFs expression, we examined the IRFs protein expression 12 h after serum depletion. As shown in Fig. 3, consistent with the mRNA expression of IRF-1 (Fig. 1 A), we observed that IRF-1 protein was up-regulated after serum depletion and IRF-1 antisense pretreatment inhibited increased IRF-1 expression, whereas IRF-1 sense and IRF-2 sense and antisense oligonucleotides did not influence the IRF-1 expression. On the other hand, IRF-2 protein level was not changed 12 h after serum depletion, although IRF-2 mRNA transiently decreased 3 h after serum removal and returned to basal level rapidly. IRF-2 antisense oligonucleotide treatment decreased the expression of IRF-2. Next we examined the effects of IRFs antisense oligonucleotides on AT2 receptor expression and observed that IRF-2 antisense enhanced AT2 receptor binding and IRF-1 antisense oligonucleotide attenuated AT2 receptor binding in confluent serum-fed R3T3 cells (Fig. 2 C). We demonstrated further that IRF-1 antisense oligonucleotide pretreatment abolished the up-regulation of AT2 receptor after serum removal (Fig.2 C), demonstrating that the increase in IRF-1 mediated the up-regulation of AT2 receptor expression. In the serum-depleted state, IRF-2 antisense treatment increased the AT2 receptor density. These data suggest that increased IRF-1 and decreased IRF-2 after serum removal may exert synergistic effects on AT2 receptor up-regulation. To test the possibility that the increased expression of IRF-1 in R3T3 cells after serum growth factor removal contributes to the induction of apoptosis, we treated the R3T3 cells with antisense oligonucleotides for IRF-1 or IRF-2 1 day before serum starvation. As shown in Fig. 4, serum depletion induced the internucleosomal cleavage of DNA, which resulted in the generation of DNA fragments of multiples of ∼180 base pairs (the size of a nucleosome): a hallmark of apoptosis. IRF-1 antisense oligonucleotide treatment inhibited DNA fragmentation in serum-starved R3T3 cells, whereas IRF-1 antisense did not affect DNA fragmentation in serum-fed R3T3 cells, suggesting that the increase of IRF-1 after serum depletion plays a critical role in the induction of apoptosis. On the other hand, IRF-2 antisense oligonucleotide did not influence the development of apoptosis. However, we cannot exclude the possibility that the transient decrease of IRF-2 mRNA after serum starvation as shown in Fig. 1 enhanced the IRF-1 effect and consequently contributed to the process of apoptosis as well. In addition to the DNA fragmentation, we also examined the morphological changes of apoptosis. As shown in Fig. 5, serum depletion caused apoptotic morphological changes in R3T3 cells. IRF-1 antisense oligonucleotide pretreatment attenuated the induction of apoptosis after serum growth factor removal, consistent with the results of DNA laddering.Figure 5Morphological changes of apoptosis in R3T3 cells. The chromatin binding dye Hoechst 33342 stains cells with intact plasma membrane and propidium iodide stains cells with loss of plasma membrane integrity. A, R3T3 cells were maintained in 10% fetal calf serum. B, serum was depleted from the medium, and typical morphological changes of the apoptosis were observed 2 days after serum starvation, showing nuclear condensation (D) and fragmentation (E and F).C, morphological apoptotic changes were attenuated by pretreatment of IRF-1 antisense oligonucleotide.View Large Image Figure ViewerDownload (PPT) As shown in Figs. 6and 7, Ang II (10−7m) treatment further enhanced DNA fragmentation after serum removal, and this effect was blocked by the specific AT2 receptor antagonist, PD123319 (Fig. 6, A and B). The effect of Ang II on the induction of apoptosis was observed both in serum-fed and serum-starved cells; however, the effect of Ang II was significantly greater in serum-starved cells. Next we examined the effects of IRFs on AT2 receptor-mediated DNA fragmentation after serum removal (Fig. 7, A and B). Antisense oligonucleotides for IRF-1 and IRF-2 were transfected into R3T3 cells; serum was removed from the culture medium 1 day after antisense transfection. The cells were then stimulated with Ang II (10−7m). Three days after Ang II stimulation, DNA fragmentation was measured. IRF-1 antisense pretreatment inhibited Ang II-mediated DNA fragmentation, whereas IRF-2 antisense pretreatment further enhanced the effect of Ang II as a result of an increased expression of AT2 receptor as shown in Fig. 2 C. Taken together these results suggest that the up-regulated AT2 receptor density, as a result of the changes in IRF-1 and IRF-2, enhances the AT2 receptor-mediated apoptosis after serum depletion.Figure 7Attenuation of AT2receptor-mediated apoptosis by IRF-1 antisense oligonucleotide.Confluent R3T3 cells were treated with antisense (AS) and sense (S) oligonucleotides for IRF-1 and IRF-2. One day after oligonucleotide transfection, serum was removed and cells were incubated with or without Ang II (10−7m) for 3 days. Quantitative analysis was performed by calculating the radioactivity incorporated into the low molecular weight fraction (<20 kb) divided by the radioactivity of serum-fed, non-treated cells (n = 4 each condition, mean ± S.D.). * showsp < 0.01 versus serum-starved, non-Ang II-treated cells. † shows p < 0.01 versusserum-starved, Ang II-treated cells.View Large Image Figure ViewerDownload (PPT) The antigrowth effects of AT2 receptor on vascular smooth muscle cells (9Nakajima, M., Hutchinson, H., Fujinaga, M., Hayashida, W., Morishita, R., Zhang, L., Horiuchi, M., Pratt, R. E., and Dzau, V. J. (1997)Proc. Natl. Acad. Sci. U. S. A., in press.Google Scholar) and endothelial cells (10Stoll M. Steckelings M. Paul M. Bottari S.P. Metzger R. Unger T. J. Clin. Invest. 1995; 95: 651-65712Google Scholar) have been reported recently. The highly abundant expression of AT2 receptor during embryonic and neonatal growth, the rapid disappearance after birth (4Grady E.F. Sechi L.A. Griffin C.A. Schambelan M. Kalinyak J.E. J. Clin. Invest. 1991; 88: 921-933Google Scholar, 6Tsutsumi K. Stromberg C. Viswanathan M. Saavedra J.M. Endocrinology. 1991; 129: 1075-1082Google Scholar), and the up-regulation of AT2 receptor in myocardial infarction (27Nio Y. Matsubara H. Murasawa S. Kanasaki M. Inada M. J. Clin. Invest. 1995; 95: 46-54Google Scholar), cardiac hypertrophy (28Lopez J. Lorell B.H. Ingelfinger J.R. Weinberg E.O. Schunkert H. Diamant D. Tang S.-H. Am. J. Physiol. 1994; 36: H844-H852Google Scholar), and skin wound (29Viswanathan M. Saavedra J.M. Peptides. 1992; 13: 783-786Google Scholar) suggest that this receptor is closely involved with growth, development, and/or differentiation. Dudley et al. (14Dudley D.T. Summerfelt R.M. Regul. Pept. 1993; 44: 199-206Google Scholar) reported that the addition of growth factors, such as basic fibroblast growth factor or serum, to quiescent R3T3 cells caused a rapid decrease in the number of surface AT2 receptor sites. Leung et al. (30Leung K.H. Roscoe W.A. Smith R.D. Timmermans P.B.W.M. Chiu A.T. Eur. J. Pharmacol. 1992; 227: 63-70Google Scholar) reported that nerve growth factor treatment decreased AT2 receptor expression in PC12W cells. Kambayashiet al. (31Kambayashi Y. Bardhan S. Inagami T. Biochem. Biophys. Res. Commun. 1993; 194: 478-482Google Scholar) also reported that the expression of AT2 receptor in cultured vascular smooth muscle cells was down-regulated by the addition of growth factors such as platelet-derived growth factor-BB, epidermal growth factor, and endothelin-1. Recently we have reported that this receptor induces apoptosis in PC12W cells by antagonizing the effect of nerve growth factor, and that apoptotic PC12W cells exhibited up-regulated AT2 receptor density (11Yamada T. Horiuchi M. Dzau V.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 156-160Google Scholar). Moreover, we demonstrated that Ang II enhanced apoptosis in serum-starved R3T3 cells, along with the increased concentration of the AT2 receptor. These studies suggest that the expression of the AT2 receptor is inversely related to cell growth and is positively correlated with apoptosis. Consistent with our previous reports (11Yamada T. Horiuchi M. Dzau V.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 156-160Google Scholar), we also demonstrated in this paper that AT2 receptor induces apoptosis in R3T3 cells; however, the "genetic evidence" that AT2 receptor induces apoptosis still remains to be proved. To examine the molecular mechanism of AT2 receptor up-regulation in apoptosis, we studied R3T3 cells that express very low level of AT2 receptor in the growing state and abundant AT2 receptor after reaching the confluent state (14Dudley D.T. Summerfelt R.M. Regul. Pept. 1993; 44: 199-206Google Scholar). To investigate the growth-regulated AT2receptor expression, we first cloned mouse AT2 receptor genomic DNA and studied its promoter function in R3T3 cells (15Horiuchi M. Koike G. Yamada T. Mukoyama M. Nakajima M. Dzau V.J. J. Biol. Chem. 1995; 270: 20225-20230Google Scholar). We identified IRF binding motif (IRF-Es) in the promoter region of AT2 receptor gene and demonstrated that IRF-2 attenuated the AT2 receptor expression in both growing and confluent R3T3 cells, whereas IRF-1 enhanced AT2 receptor expression in the confluent cells only. Consistent with this result, gel mobility shift assay demonstrated that growing R3T3 cells exhibited only IRF-2 binding, whereas confluent cells exhibited both IRF-1 and IRF-2 bindings. In this paper, we demonstrated further that, in confluent R3T3 cells, the removal of serum increased IRF-1 expression but caused a rapid and transient decrease in IRF-2 mRNA expression. These changes of IRFs were associated with an up-regulation of AT2 receptor and the enhancement of AT2 receptor-mediated apoptosis. These results suggest that the IRFs play an important role in the up-regulation of this receptor associated with apoptosis. IRF-1 and IRF-2 are structurally related, and they recognize the same DNA sequence elements AAGTGA motif or G(A)AAAG/CT/CGAAAG/CT/C (16Harada H. Fujita T. Miyamoto M. Kimura Y. Maruyama M. Furia A. Miyata T. Taniguchi T. Cell. 1989; 58: 729-739Google Scholar, 17Tanaka N. Kawakami T. Taniguchi T. Mol. Cell. Biol. 1993; 13: 4531-4538Google Scholar). Gene transfection studies have demonstrated that these two factors are mutually antagonistic; IRF-1 activates transcription, whereas IRF-2 inhibits it (16Harada H. Fujita T. Miyamoto M. Kimura Y. Maruyama M. Furia A. Miyata T. Taniguchi T. Cell. 1989; 58: 729-739Google Scholar, 19Harada H. Willison K. Sakakibara J. Miyamoto M. Fujita T. Taniguchi T. Cell. 1990; 63: 303-312Google Scholar). Harada et al. (23Harada H. Kitagawa M. Tanaka N. Yamamoto H. Harada K. Ishihara M. Taniguchi T. Science. 1993; 259: 971-974Google Scholar) have shown that the IRF-1/IRF-2 expression ratio oscillates throughout the cell cycle of NIH 3T3 cells, with IRF-1 expression at its highest when cells are growth-arrested owing to serum starvation and with IRF-2 being more abundant in growing cells. IRF-1 manifests antiproliferative properties (20Yamada G. Ogawa M. Akagi K. Miyamoto H. Nakano N. Itoh S. Miyazaki J. Nishikawa S. Yamamura K. Taniguchi T. Proc. Natl. Acad. Sci. U. S. A. 1990; 88: 532-536Google Scholar, 22Kirchhoff S. Schaper F. Hauser H. Nucleic Acids Res. 1993; 21: 2881-2889Google Scholar), whereas overexpression of the repressor IRF-2 leads to cell transformation and increased tumorigenicity, and this phenotype can be reverted by the concomitant overexpression of IRF-1 (23Harada H. Kitagawa M. Tanaka N. Yamamoto H. Harada K. Ishihara M. Taniguchi T. Science. 1993; 259: 971-974Google Scholar). Accordingly, we speculate further that the changes of IRF-1 and IRF-2 after serum removal are closely linked to the induction of apoptosis in R3T3 cells. Indeed our data demonstrated an increased IRF-1 expression and transiently decreased IRF-2 expression during apoptosis induced by serum removal. Several lines of evidence has shown that the expression of oncogenes can sensitize cells to undergo apoptosis, particularly under conditions of low serum concentration or high cell density (32Evan G.I. Wyllie A.H. Gilbert C.S. Littlewood T.D. Land H. Brooks M. Waters C.M. Penn L.Z. Hancock D.C. Cell. 1992; 69: 119-128Google Scholar, 33Rao L. Debbas M. Sabbatini P. Hockenbery D. Korsmeyer S. White E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7742-7746Google Scholar). It has been demonstrated that IRF-1 and IRF-2 have antioncogenic and oncogenic activities, respectively. Deletion or inactivation of the IRF-1 gene at one or both alleles have been detected in leukemia and myelodysplastic syndromes (34Willman G.L. Sever C.E. Pallavicini M.G. Harada H. Tanaka N. Slovak M.L. Yamamoto H. Harada K. Meeker T.C. List A.F. Taniguchi T. Science. 1993; 259: 968-971Google Scholar). Tanaka et al. (35Tanaka N. Ishihara M. Kitagawa M. Harada H. Kimura T. Matsuyama T. Lamphier M.S. Aizawa S. Mak T.W. Taniguchi T. Cell. 1994; 77: 829-839Google Scholar) reported that IRF-1 may be a critical determinant of oncogene-induced cell transformation or apoptosis in mouse embryonic fibroblasts (EFs). They demonstrated that Ras signaling, under conditions of low serum or at high cell density or following treatment by anticancer drugs or ionizing radiation, induces the death of EFs derived from wild type mice and from mice with a null mutation in the IRF-2 gene (IRF-2−/− mice), but not of EFs from IRF-1−/− and double knockout mice. These data demonstrate that the absence of IRF-1 alone is sufficient to prevent Ras-induced apoptosis. Tamura et al. (36Tamura T. Ishihara M. Lamphier M.S. Tanaka N. Oishi I. Aizawa S. Matsuyama T. Mak T.W. Taki S. Taniguchi T. Nature. 1995; 376: 596-599Google Scholar) also showed that the mitogen induction of the interleukin-1β-converting enzyme, mammalian homologue of the Caenorhabditis elegance cell death gene ced-3, is IRF-1-dependent. These studies are consistent with our present results and support further that IRF-1 is an unique transcription factor that functions as an apoptotic inducer. IRF-1 and IRF-2 are originally identified as regulators of type I interferon system. DNA sequences recognized by IRFs have been also found in the regulatory regions in a number of interferon-inducible genes (16Harada H. Fujita T. Miyamoto M. Kimura Y. Maruyama M. Furia A. Miyata T. Taniguchi T. Cell. 1989; 58: 729-739Google Scholar, 17Tanaka N. Kawakami T. Taniguchi T. Mol. Cell. Biol. 1993; 13: 4531-4538Google Scholar, 18Fujita T. Kinura Y. Miyamoto M. Barsoumian E.L. Taniguchi T. Nature. 1989; 337: 270-272Google Scholar, 19Harada H. Willison K. Sakakibara J. Miyamoto M. Fujita T. Taniguchi T. Cell. 1990; 63: 303-312Google Scholar, 37Fujita T. Sakakibara J. Sudu Y. Miyamoto M. Kimura Y. Taniguchi T. EMBO J. 1988; 7: 3397-3405Google Scholar, 38Pine R. Levy D.E. Reich N. Darnell Jr., J.E. Mol. Cell. Biol. 1990; 10: 2448-2457Google Scholar, 39Reis L.F. harada H. Wolchok J.D. Taniguchi T. Vilcek J. EMBO J. 1992; 11: 185-193Google Scholar). IRF binding consensus element was also identified in inducible nitric oxide synthase (iNOS) gene promoter region, and IRF-1 is essential for iNOS activation in murine macrophages (40Kamijo R. Harada H. Matsuyama T. Bosland M. Gerecitano J. Shapiro D. Le J. Koh S.I. Kimura T. Green S.J. Mak T.W. Taniguchi T. Vilcek J. Science. 1994; 263: 1612-1615Google Scholar). Recently we reported that NO donor molecules,S-nitroso-N-acetylpenicillamine or sodium nitroprusside, induced apoptosis in cultured rabbit vascular smooth muscle cells (41Pallman M. Yamada M. Horiuchi M. Gibbons G.H. Circ. Res. 1996; 79: 748-756Google Scholar). In this study, we observed that an increase in the ratio of IRF-1/IRF-2 after serum starvation mediated the up-regulation of AT2 receptor, resulting in the enhancement of Ang II-mediated apoptosis. Our data showed that the blockade of IRF-1 expression resulted in the simultaneous reduction in AT2receptor expression and in apoptosis, whereas blockade of IRF-2 expression led to an increase in AT2 receptor expression and enhanced apoptosis. Taken together these data support the notion that AT2 receptor is one of the target genes of the IRFs system. We propose further that IRF-regulated gene products such AT2 receptor and iNOS may play important mediating roles in IRF-regulated apoptosis. We gratefully acknowledge Dr. M. de Gasparo for CGP42112A, Dr. D. Dudley for R3T3 cells, and M. Hing for secretarial assistance.