Contribution of Extracellular Signal-Regulated Kinase to Angiotensin II–Induced Transforming Growth Factor-β1 Expression in Vascular Smooth Muscle Cells
Abstract—We have previously demonstrated that angiotensin II (Ang II) contributes to the increase in aortic transforming growth factor-β1 (TGF-β1) mRNA levels in hypertensive rats. However, the molecular mechanism whereby Ang II promotes TGF-β1 expression in vascular smooth muscle cells (VSMCs) is poorly understood. In this study, we examined the role of extracellular signal–regulated kinase (ERK) in Ang II–mediated TGF-β1 expression in VSMCs and the role of Ang II in aortic ERK activity of stroke-prone spontaneously hypertensive rats. Treatment of quiescent VSMCs with 100 nmol/L Ang II induced rapid phosphorylation and activation of ERK1 and ERK2 with a peak at 5 minutes followed by an increase in activator protein-1 (AP-1) DNA binding activity, as shown by gel mobility shift assay. An increase in TGF-β1 mRNA was shown by Northern blot analysis. Treatment of VSMCs with PD98059, a specific inhibitor of the ERK pathway, attenuated both the activation of AP-1 and the increase in TGF-β1 mRNA induced by Ang II. Inhibition of Ang II–induced AP-1 activation with c-fos antisense oligodeoxynucleotide led to a significant reduction of TGF-β1 mRNA in VSMCs. Furthermore, in vivo treatment of stroke-prone spontaneously hypertensive rats with losartan, an Ang II type 1 receptor antagonist, decreased aortic ERK activity. Thus, we show that ERK, through AP-1 activation, is involved in Ang II–induced TGF-β1 mRNA expression in VSMCs and suggest that ERK may participate in vascular remodeling of hypertension. However, it remains to be determined whether the increase in TGF-β1 mRNA leads to the increase in its active protein.
- muscle, smooth, vascular
- angiotensin II
- transforming growth factor beta
- protein kinases
- oligonucleotides, antisense
Extracellular signal-regulated kinase (ERK), protein Ser/Thr kinase, is a subgroup of the mitogen-activated protein kinase (MAPK) family. Accumulating in vitro evidence indicates that ERK is activated by various extracellular stimuli, mostly vasoactive peptides and growth factors, and plays an important role in cell growth and the regulation of gene expression.1 2 We have recently reported that ERK activity is progressively increased in the aorta of hypertensive rats compared with normotensive rats.3 However, the regulatory mechanism and the role of ERK in vascular diseases remain unclear.
A growing body of evidence supports the finding that angiotensin II (Ang II) is a critical mediator of vascular hypertrophy and neointimal hyperplasia in various vascular diseases.4 5 6 7 8 Ang II stimulates the activation of ERK in cultured vascular smooth muscle cells (VSMCs).9 10 This ERK activation was recently shown to be responsible for increased protein synthesis in VSMCs by Ang II.11 However, little is known about the pathophysiological significance of Ang II-induced ERK activation in VSMCs.
Transforming growth factor-β1 (TGF-β1), a growth factor that stimulates cell hypertrophy and extracellular matrix (ECM) production,12 13 has been shown to be involved in Ang II-mediated hypertrophy of VSMCs.14 In vivo administration of TGF-β1 enhanced intimal thickening in balloon injured arteries.15 Administration of specific antibodies against TGF-β1 in vivo suppresses intimal hyperplasia in the rat balloon-injured artery.16 Thus, TGF-β1 plays a key role in vascular remodeling in vivo. Furthermore, we have previously reported that aortic TGF-β1 mRNA is increased in the aorta of hypertensive rats and is accompanied by ECM accumulation17 18 and that treatment of hypertensive rats with Ang II type 1 receptor (AT1) antagonist significantly decreases aortic TGF-β1 mRNA levels. Thus, our reports suggest that TGF-β1 may be implicated in the mechanism of Ang II–mediated vascular injury. However, the molecular mechanism underlying the induction of vascular TGF-β1 by Ang II is poorly understood.
In the present study, we examined the possible involvement of ERK in Ang II–induced TGF-β1 expression in VSMCs and investigated the in vivo role of Ang II in vascular ERK activity in hypertensive rats. We obtained the first evidence that ERK activation, mediated by activator protein-1 (AP-1), is responsible for Ang II–induced TGF-β1 expression in VSMCs and suggested that Ang II might contribute to the increased aortic ERK activity of hypertensive rats.
Rabbit anti-phospho-ACTIVE MAP kinase antibodies were purchased from Promega. Anti-p44 ERK (C-16), anti-p42 ERK (C-14), anti-c-Fos (K-25), and anti-c-Jun (N) antibodies were all purchased from Santa Cruz Biotechnology, Inc. The MAPK kinase (MEK) inhibitor PD98059 was purchased from New England Biolab. Tyrphostin AG1478 and A63 were purchased from Calbiochem. CV-11974, losartan, and PD123319 were kind gifts from Takeda Chemical Industries, Ltd (Osaka, Japan), Dupont Merck (Wilmington, Del), and Parke-Davis (Ann Arbor, Mich), respectively.
Rat VSMCs were isolated from thoracic aorta of male Sprague-Dawley rats by using the collagenase digestion method and were maintained in DMEM with 10% fetal bovine serum (FBS) and penicillin/streptomycin. All cell cultures were kept in a humidified 5% CO2/95% air incubator at 37°C and were used from passages 4 to 7. For all experiments, VSMCs were grown to 70% confluence and then made quiescent by incubation with 0.1% FBS for 48 hours.
In-Gel Kinase Assay
After treatment, the cells were washed with precooled PBS and lysed with the lysis buffer (20 mmol/L HEPES, pH 7.2, 25 mmol/L NaCl, 2 mmol/L EGTA, 50 mmol/L NaF, 1 mmol/L Na3VO4, 25 mmol/L β-glycerophosphate, 0.2 mmol/L DTT, 1 mmol/L PMSF, 60 μg/mL aprotinin, and 0.1% Triton X-100). After a brief sonication, the cells were centrifuged, and the supernatants were stored at −80°C until use. ERK activity was determined by in-gel kinase assay, as previously described.19 To evaluate kinase activities, we digitized autoradiograms and measured their densities by using a bioimaging analyzer (BAS-2000; Fuji Photo Film Co).
Western Blot Analysis
After treatment, the cells were washed with precooled PBS, and lysed with the same buffer as used in the in-gel kinase assay. Western blot analysis was carried out using anti-p42ERK, anti-p44ERK, or anti–phospho-ERK antibodies, as previously described.20 Immune complexes were visualized by using the enhanced chemiluminescence method (Amersham). We measured their density with the use of the public domain National Institutes of Health IMAGE program.
Liposomal Transfection With c-fos Antisense Oligodeoxynucleotide
The antisense phosphorothioate oligodeoxynucleotide (ODN) (20-mer [5′-CCATGATGTTCTCGGGTTTC-3′]) was directed against the initiation site of c-fos mRNA. Corresponding sense ODN was used as control. Transfection with 0.4 μmol/L ODN was performed in serum-free DMEM with 8 μg/mL Lipofectamine reagent (Gibco BRL) for 6 hours at 37°C in 5% CO2/95% air. Medium was then replaced with Lipofectamine-free DMEM containing 0.1% FBS and the same concentration of ODN, and VSMCs were cultured further for 42 hours in a 5% CO2/95% air incubator at 37°C.
Electrophoretic Mobility Shift Assay
Electrophoretic gel mobility shift assays (EMSAs) of nuclear protein extracts (3 μg of protein) were performed, as described in detail previously.19 The double-stranded consensus oligonucleotide sequence of AP-1 used was 5′-CGCTTGATGACTCAGCCGGAA-3′. To demonstrate the specificity of DNA protein binding, the reactions were performed in the presence of nonlabeled consensus oligonucleotide competitor or nonlabeled mutant oligonucleotide competitor (5′-CGCTTGATGACTTGGCCGGAA-3′). Furthermore, supershift assay was performed with rabbit polyclonal IgG against c-Fos or c-Jun.
RNA Preparation and Northern Blot Analysis
All procedures were performed, as previously described.17 Total RNA (each 7 μg) was isolated by the guanidium thiocyanate-phenol-chloroform method with minor modification. The cDNA probes used were rat TGF-β1 cDNA21 and rat GAPDH.22 Densities of an individual mRNA were measured by using a bioimaging analyzer and were divided by that of GAPDH mRNA for the correction of the difference between RNA loading and transfer to a nylon membrane.
In Vivo Experiments in Hypertensive Rats
All procedures were in accordance with institutional guidelines for animal research of Osaka City University Medical School. Male 20-week-old stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto rats (WKY) were purchased from Japan SLC (Shizuoka, Japan). SHRSP were orally given vehicle or 3 doses of the AT1 antagonist losartan (6, 30, or 60 mg/kg) by gastric gavage once a day for 2 weeks. Losartan was suspended with 0.5% carboxymethyl cellulose. Control WKY rats were given 0.5% carboxymethyl cellulose for the same period. Systolic blood pressure was measured by the tail-cuff method. After 2 weeks of treatment, SHRSP and WKY were decapitated and thoracic aortas were immediately excised, frozen in liquid nitrogen, and stored at −80°C until use. For in-gel kinase assay, aortic protein extracts were prepared as previously described.3
To examine further whether the effect of AT1 antagonist on aortic ERK activity is due to its direct inhibition of AT1 or to its hypotensive effect, we performed another experiment. A calcium channel blocker, nifedipine, at a dose of 15 mg/kg, was given to SHRSP by gastric gavage 3 times a day (daily dose of 45 mg · kg−1 · d−1). After the treatment, aortic ERK activity was measured in the same manner as described above.
Data are expressed as mean±SEM. Statistical significance was determined by ANOVA and Duncan’s multiple range test. Because the values of kinase activities sometimes showed heteroscedasticity, logarithmic transformation was made for data before analysis. Differences were considered statistically significant at a value of P<0.05.
Ang II Induces Phosphorylation and Activation of ERK in VSMCs
After quiescent VSMCs were stimulated with Ang II (100 nmol/L), we measured the phosphorylation of ERK by Western blot analysis and the activation of ERK by in-gel kinase assay. As shown in Figure 1A⇓, in-gel kinase assay showed that ERK1 (44 kDa) and ERK2 (42 kDa) activities rapidly increased, peaked at 5 minutes, and then gradually declined to basal level. The increase in these ERK activities was associated with the increase in phosphorylated ERK. However, total protein levels of these ERK were not affected by Ang II. Figure 1B⇓ shows the effects of pretreatment with several inhibitors on ERK activation by Ang II. The increase in ERK activities by Ang II was inhibited by AT1 antagonist, CV-11974, but not by Ang II type 2 receptor (AT2) antagonist, PD123319, which indicates that the activation of ERK was mediated through AT1. Pretreatment with PD98059, which selectively blocks the activity of ERK pathway at the level of MAPK kinase (MEK), decreased Ang II–induced ERK activities in a dose-dependent manner. In addition, treatment with an inhibitor of tyrosine phosphorylation of epithelial growth factor (EGF) receptor, tyrphostin AG1478, also inhibited ERK activation by Ang II. On the other hand, inactive tyrphostin analogue, A63, failed to inhibit ERK activity.
Ang II Increases AP-1 DNA Binding Activity and TGF-β1 mRNA in VSMCs
We examined the effects of Ang II on AP-1 DNA binding activity in VSMCs by using EMSA. As shown in Figure 2A⇓, the band indicated by the bracket was decreased by addition of unlabeled AP-1 consensus oligonucleotide in a dose-dependent fashion but was not decreased by excess amounts of unlabeled mutant AP-1 oligonucleotide, which indicates that this band was a specific binding for AP-1. The AP-1 binding complex was supershifted by addition of either anti-c-Fos or anti-c-Jun antibodies. Figure 2B⇓ shows that AP-1 DNA binding activity was increased by 2.4-fold at 2 hours after Ang II stimulation, peaked at 4 hours (2.7-fold) and had declined almost to basal level after 24 hours. As shown by Northern blot analysis in Figure 2C⇓, TGF-β1 mRNA increased by Ang II stimulation, reached the peak at 4 to 6 hours (1.4-fold) and gradually returned to the basal level within 24 hours.
Effects of PD98059 on AP-1 Binding Activity and TGF-β1 Expression in VSMCs
As shown in Figures 3A⇓ and 3B⇓, the increase in both AP-1 activity and TGF-β1 mRNA by Ang II was inhibited by pretreatment with CV-11974 but not with PD123319, which indicates that AP-1 activation and TGF-β1 expression by Ang II were mediated via AT1 in VSMCs. Pretreatment of quiescent VSMC with PD98059 significantly decreased Ang II–mediated AP-1 binding activity and also attenuated TGF-β1 mRNA in VSMCs. We also examined whether the blockade of ERK activity by AG1478 would inhibit Ang II–mediated AP-1 activity and TGF-β1 expression in VSMCs. We showed that treatment of VSMCs with AG1478 prevented the increase in both AP-1 activity and TGF-β1 mRNA induced by Ang II.
Effects of c-fos Antisense ODN on AP-1 Binding Activity and TGF-β1 Expression in VSMCs
To determine whether Ang II–induced activation of AP-1 was responsible for the increased TGF-β1 mRNA, we examined the effect of c-fos antisense ODN on TGF-β1 expression. Treatment with antisense c-fos ODN significantly reduced Ang II–induced AP-1 binding activity in VSMCs, whereas sense sequence ODN had no effect on AP-1 activity (Figure 4A⇓). As shown in Figure 4B⇓, the Ang II–induced increase in TGF-β1 mRNA was also significantly decreased by c-fos antisense ODN. Sense ODN did not affect TGF-β1 expression.
Effects of AT1 Antagonist on Aortic ERK Activities of Hypertensive Rats
Blood pressure of vehicle-treated 22-week-old SHRSP (234±8 mm Hg) was higher than that of age-matched WKY (140±3 mm Hg)(P<0.01). Treatment with losartan (6, 30, and 60 mg/kg) for 2 weeks significantly decreased blood pressure of SHRSP (195±2, 154±2, and 135±2 mm Hg, respectively). As shown in Figure 5⇓, aortic ERK1 and ERK2 activities in SHRSP were 2.6- and 2.0-fold higher, respectively, than those in WKY (P<0.01). Treatment of SHRSP with the AT1 antagonist losartan decreased aortic ERK1 and ERK2 activities in a dose-dependent manner. Losartan at 60 mg · kg−1 · d −1 completely normalized the increase in aortic ERK1 and ERK2 activities of SHRSP to the levels of WKY (Figure 5⇓).
To examine whether the decrease in aortic ERK activity by losartan was due to the inhibition of AT1 or to the hypotensive effect of losartan, we examined the effects of nifedipine (45 mg · kg−1 · d−1) on aortic ERK of SHRSP in another experiment. Blood pressure of SHRSP (230±7 mm Hg) was completely normalized by nifedipine (130±5 mm Hg; P<0.01), similar to the above-mentioned hypotensive effect of 60 mg · kg−1 · d−1 of losartan. When the mean values in WKY were represented as 1, aortic ERK1 and ERK2 activities were 1.00±0.08 and 1.00±0.07, respectively, in WKY(n=5); 2.15±0.09 and 1.89±0.12, respectively, in vehicle-treated SHRSP(n=6); and 1.61±0.06 and 1.52±0.05, respectively, in nifedipine-treated SHRSP (n=6). Both ERK1 and ERK2 activities in nifedipine-treated SHRSP were significantly higher than those in WKY (P<0.01). Thus, in contrast to the complete normalization of aortic ERK1 and ERK2 by losartan (Figure 5⇑), normalization of blood pressure by nifedipine treatment only partially suppressed the increase in aortic ERK1 and ERK2 activities of SHRSP.
Accumulating evidence supports the finding that Ang II via AT1 plays a key role in vascular hypertrophy and in remodeling of various vascular diseases, including hypertension, diabetes mellitus, and balloon injury.7 8 23 However, the molecular mechanism responsible for Ang II–induced vascular injury remains to be determined. In the present study, we provided the first evidence that ERK activation by Ang II, through the activation of AP-1, contributes to TGF-β1 induction in VSMCs, and the results of our study suggested that the ERK activation might be involved in vascular remodeling of hypertension. Thus, our present study provided a new insight into the molecular mechanism of Ang II–induced vascular remodeling.
ERK, a member of the MAPK subfamily, plays an important role in cell growth and the regulation of gene expression.1 2 Recently, it has been shown that the inhibition of Ang II–induced activation of the ERK pathway with PD98059, a MEK inhibitor, leads to a decrease in protein synthesis in VSMCs,11 indicating that ERK is involved in Ang II–induced VSMC hypertrophy. However, the role of ERK in vascular remodeling in vivo is poorly understood. More recently, we have shown that aortic ERK activity is progressively increased in hypertensive rats versus normotensive rats.3 We have previously also demonstrated that aortic TGF-β1 mRNA is enhanced in hypertensive rats.17 18 Our in vivo findings,3 17 18 taken together with previous in vitro findings that VSMC hypertrophy by Ang II in vitro is mediated by TGF-β1 induction and its subsequent autocrine action,14 encouraged us to examine the possible role of ERK in TGF-β1 expression by Ang II. Of note are the observations that the inhibition of ERK pathway with PD98059 resulted in the significant suppression of Ang II-induced TGF-β1 induction in VSMCs, providing the first evidence that the ERK pathway plays an important role in TGF-β1 expression by Ang II in VSMCs.
Ang II is well known to induce c-fos and c-jun mRNAs rapidly in VSMCs as estimated by Northern blot analysis.24 25 However, the significance of induction of c-fos and c-jun mRNAs by Ang II in VSMCs remains to be determined. Therefore, in the present study, we examined the effect of Ang II on transcription factor AP-1 DNA binding activity by EMSA. We found that Ang II significantly increased AP-1 DNA binding activity following ERK activation and that AP-1 binding complex contained c-Fos and c-Jun proteins as shown by supershift analysis. Therefore, the increase in c-fos and c-jun mRNA by Ang II seems to play an important role in the activation of transcription factor AP-1 in VSMCs.
In the present study, we also found that the inhibition of ERK cascade with PD98059 led to the decrease in AP-1 DNA binding activity. This observation, taken together with the fact that ERK induces c-fos mRNA via the phosphorylation of TCF/Elk-1,26 27 supports the thesis that ERK is involved in the increased AP-1 DNA binding activity. Furthermore, treatment of VSMCs with c-fos antisense ODN, which resulted in the significant reduction of AP-1 DNA binding activity, significantly decreased TGF-β1 mRNA. These findings, together with the fact that the promoter region of TGF-β1 gene contains AP-1 responsive element,28 indicate that c-fos plays a key role in TGF-β1 expression by Ang II.
Recently, it has been reported that ERK activation via AT1 in VSMCs is mediated by EGF receptor tyrosine phosphorylation.29 Therefore, to examine the role of EGF receptor in Ang II–induced TGF-β1 expression, we examined the effect of a specific EGF receptor tyrosine kinase inhibitor (AG1478) on TGF-β1 expression. AG1478 significantly inhibited ERK activation by Ang II in VSMCs; in contrast, AG63 (inactive tyrphostin analogue) showed no effect, which agrees with a previous report.29 As in the case of treatment with PD98059, the inhibition of ERK activation by AG1478 caused the inhibition of TGF-β1 expression, thereby indicating that ERK activation by Ang II via EGF receptor induces TGF-β1 expression (Figure 6⇓).
In the present study, to examine the role of Ang II in the increased aortic ERK activity of SHRSP, we compared the effect of AT1 antagonist to that of nifedipine on aortic ERK activity of SHRSP. We obtained the first evidence supporting the proposition that Ang II via AT1 is partially responsible for the enhanced aortic ERK activity in SHRSP. Furthermore, we have previously reported that treatment with an AT1 antagonist decreased aortic TGF-β1 expression in SHRSP. This finding, accompanied by the decrease in ECM components, supports the important role of AT1 in the enhanced aortic TGF-β1 mRNA of SHRSP.17 These in vivo findings, taken together with our present in vitro findings on VSMC, suggest that increased aortic ERK activity may be responsible for the increased aortic TGF-β1 expression in SHRSP. However, the present study did not permit us to determine whether the chronic increase of aortic ERK activity in SHRSP can be attributed to the enhancement of the upstream signaling cascade, the increase in ERK protein, or the decreased activity of phosphatases.
In conclusion, our present study provides the first evidence that Ang II–induced ERK activation participates in TGF-β1 expression in VSMCs and suggests that ERK may, in part, be responsible for vascular remodeling by Ang II in vivo, through TGF-β1 expression. However, because the regulation of TGF-β1 could occur at levels other than at its transcription, further study is needed to elucidate whether the increased TGF-β1 mRNA by Ang II leads to the increase in its active protein.
This work was supported in part by grants for scientific research 09670101 and 09470527 from the Ministry of Education, Science, Sports, and Culture of Japan.
- Received September 14, 1998.
- Revision received October 16, 1998.
- Accepted March 8, 1999.
Force T, Bonventre JV. Growth factors and mitogen-activated protein kinases. Hypertension. 1998;31:152–161.
Geisterfer AA, Peach MJ, Owens GK. Angiotensin II induces hypertrophy, not hyperplasia, of cultured rat aortic smooth muscle cells. Circ Res. 1988;62:749–756.
Berk BC, Vekshtein V, Gordon HM, Tsuda T. Angiotensin II–stimulated protein synthesis in cultured vascular smooth muscle cells. Hypertension. 1989;13:305–314.
Kim S, Kawamura M, Wanibuchi H, Ohta K, Hamaguchi A, Omura T, Yukimura T, Miura K, Iwao H. Angiotensin II type 1 receptor blockade inhibits the expression of immediate-early genes and fibronectin in rat injured artery. Circulation. 1995;92:88–95.
Kim S, Wanibuchi H, Hamaguchi A, Miura K, Yamanaka S, Iwao H. Angiotensin blockade improves cardiac and renal complications of type II diabetic rats. Hypertension. 1997;30:1054–1061.
Tsuda T, Kawahara Y, Ishida Y, Koide M, Shii K, Yokoyama M. Angiotensin II stimulates two myelin basic protein/microtubule-associated protein 2 kinases in cultured vascular smooth muscle cells. Circ Res. 1992;71:620–630.
Griendling KK, Ushio Fukai M, Lassegue B, Alexander RW. Angiotensin II signaling in vascular smooth muscle: new concepts. Hypertension. 1997;29:366–373.
Servant MJ, Giasson E, Meloche S. Inhibition of growth factor-induced protein synthesis by a selective MEK inhibitor in aortic smooth muscle cells. J Biol Chem. 1996;271:16047–16052.
Owens GK, Geisterfer AA, Yang YW, Komoriya A. Transforming growth factor-β-induced growth inhibition and cellular hypertrophy in cultured vascular smooth muscle cells. J Cell Biol. 1988;107:771–780.
Border WA, Ruoslahti E. Transforming growth factor-β in disease: the dark side of tissue repair. J Clin Invest. 1992;90:1–7.
Gibbons GH, Pratt RE, Dzau VJ. Vascular smooth muscle cell hypertrophy vs. hyperplasia: autocrine transforming growth factor-beta 1 expression determines growth response to angiotensin II. J Clin Invest. 1992;90:456–461.
Kanzaki T, Tamura K, Takahashi K, Saito Y, Akikusa B, Oohashi H, Kasayuki N, Ueda M, Morisaki N. In vivo effect of TGF-β1: enhanced intimal thickening by administration of TGF-β1 in rabbit arteries injured with a balloon catheter. Arterioscler Thromb Vasc Biol. 1995;15:1951–1957.
Wolf YG, Rasmussen LM, Ruoslahti E. Antibodies against transforming growth factor-β1 suppress intimal hyperplasia in a rat model. J Clin Invest. 1994;93:1172–1178.
Kim S, Ohta K, Hamaguchi A, Omura T, Yukimura T, Miura K, Inada Y, Ishimura Y, Chatani F, Iwao H. Angiotensin II type I receptor antagonist inhibits the gene expression of transforming growth factor-β1 and extracellular matrix in cardiac and vascular tissues of hypertensive rats. J Pharmacol Exp Ther. 1995;273:509–515.
Hamaguchi A, Kim S, Yano M, Yamanaka S, Iwao H. Activation of glomerular mitogen-activated protein kinases in angiotensin II-mediated hypertension. J Am Soc Nephrol. 1998;9:372–380.
Kim S, Izumi Y, Yano M, Hamaguchi A, Miura K, Yamanaka S, Miyazaki H, Iwao H. Angiotensin blockade inhibits activation of mitogen-activated protein kinases in rat balloon-injured artery. Circulation. 1998;97:1731–1737.
Qian SW, Kondaidah P, Roberts AB, Sporn MB. cDNA cloning by PCR of rat transforming growth factor-β1. Nucleic Acid Res. 1990;18:3059.
Fort PH, Marty L, Piechaczyk M, Sabrouty SEL, Dani CH, Jeanteur PH, Blanchard JM. Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3-phosphate-dehydrogenase multigenic family. Nucleic Acid Res. 1985;13:1432–1442.
Dzau VJ, Gibbons GH, Pratt RE. Molecular mechanism of vascular renin-angiotensin system in myointimal hyperplasia. Hypertension. 1991;18(suppl II):II-100–II-105.
Taubman MB, Berk BC, Izumo S, Tsuda T, Alexander RW, Nadal Ginard B. Angiotensin II induces c-fos mRNA in aortic smooth muscle: role of Ca2+ mobilization and protein kinase C activation. J Biol Chem. 1989;264:526–530.
Itoh H, Mukoyama M, Pratt RE, Gibbons GH, Dzau VJ. Multiple autocrine growth factors modulate vascular smooth muscle cell growth response to angiotensin II. J Clin Invest. 1993;91:2268–2274.
Karin M. The regulation of AP-1 activity by mitogen-activated protein kinases. J Biol Chem. 1995;270:16483–16486.
Kim SJ, Glick A, Sporn MB, Roberts AB. Characterization of the promoter region of the human transforming growth factor-β1 gene. J Biol Chem. 1989;264:402–408.
Eguchi S, Numaguchi K, Iwasaki H, Matsumoto T, Yamakawa T, Utsunomiya H, Motley ED, Kawakatsu H, Owada KM, Hirata Y, Marumo F, Inagami T. Calcium-dependent epidermal growth factor receptor transactivation mediates the angiotensin II-induced mitogen-activated protein kinase activation in vascular smooth muscle cells. J Biol Chem. 1998;273:8890–8896.