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Originally published In Press as doi:10.1074/jbc.M110430200 on March 20, 2002

J. Biol. Chem., Vol. 277, Issue 21, 18710-18717, May 24, 2002
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Critical Role of cAMP-response Element-binding Protein for Angiotensin II-induced Hypertrophy of Vascular Smooth Muscle Cells*

Yuko Funakoshi, Toshihiro IchikiDagger, Kotaro Takeda, Tomotake Tokuno, Naoko Iino, and Akira Takeshita

From the Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, 812-8582 Fukuoka, Japan

Received for publication, October 30, 2001, and in revised form, January 25, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We reported previously an important role of cyclic AMP-response element (CRE) for the induction of interleukin-6 gene expression by angiotensin II (AngII). We examined signaling pathways that are responsible for AngII-induced phosphorylation of CRE-binding protein (CREB) at serine 133 that is a critical marker for the activation in rat vascular smooth muscle cells (VSMC). AngII time dependently induced phosphorylation of CREB with a peak at 5 min. The AngII-induced phosphorylation of CREB was blocked by CV11974, an AngII type I receptor antagonist, suggesting that AngII type I receptor may mediate the phosphorylation of CREB. Inhibition of extracellular signal-regulated protein kinase (ERK) by PD98059 or inhibition of p38 mitogen-activated protein kinase (MAPK) by SB203580 partially inhibited AngII-induced CREB phosphorylation. A protein kinase A inhibitor, H89, also partially suppressed AngII-induced CREB phosphorylation. Inhibition of epidermal growth factor-receptor by AG1478 suppressed the AngII-induced CREB phosphorylation as well as activation of ERK and p38MAPK. Overexpression of the dominant negative form of CREB by an adenovirus vector suppressed AngII-induced c-fos expression and incorporation of [3H]leucine to VSMC. These findings suggest that AngII may activate multiple signaling pathways involving two MAPK pathways and protein kinase A, all of which contribute to the activation of CREB. Transactivation of epidermal growth factor-receptor is also critical for AngII-induced CREB phosphorylation. Activation of CREB may be important for the regulation of gene expression and hypertrophy of VSMC induced by AngII.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Angiotensin II (AngII)1 has multiple biological functions such as vasoconstriction, induction of hypertrophy of vascular smooth muscle cells (VSMC), and secretion of growth factors and matrix components. Although two isoforms of AngII receptor, designated type 1 receptor (AT1-R) (1) and type 2 receptor (AT2-R) (2), have been cloned, most of the cardiovascular effects are ascribed to the AT1-R. Many studies have shown that AT1-R couples to a variety of protein kinase pathways. It is known that AngII activates p42/p44 extracellular signal-regulated protein kinase (ERK) (3, 4). AngII activates another class of mitogen-activated protein kinase (MAPK) such as Jun N-terminal kinase (JNK) (5) and p38 MAPK (6). It is also reported that AngII activates phosphatidylinositol 3-kinase (PI3K) and Akt/protein kinase B pathway (7). Understanding the mechanisms of AngII-stimulated signaling pathways is important because these signaling pathways activate gene transcription of immediate early genes, cytokines, and extracellular matrix components, resulting in cardiovascular remodeling.

cAMP-response element (CRE)-binding protein (CREB) (8) is a 43-kDa nuclear transcription factor belonging to the CREB/ATF family. CREB binds to the octanucleotide sequence, TGACGTCA, as a homodimer and as a heterodimer in association with other members of the CREB/ATF family (9, 10). Previous studies have demonstrated that neurotransmitters, hormones, and growth factors in different cell types can activate CREB (9, 11). Phosphorylation of a serine residue at 133 (Ser-133) is necessary for transcriptional activation of CREB. The phosphorylation of Ser-133 is mediated by a variety of kinases such as: (i) protein kinase A (PKA) (9) in response to an elevation of intracellular cAMP, (ii) calmodulin (CaM) kinases II and IV in response to an elevation in intracellular calcium (12), (iii) p90RSK2 in response to activation of a ras-dependent ERK pathway (13), (iv) MAPK-activated protein (MAPKAP) kinase 2 in response to activation of p38MAPK (14), and (v) Akt/protein kinase B by activation of PI3K (15). Phosphorylated CREB at Ser-133 recruits CREB-binding protein (CBP), which is a transcriptional coactivator, and activates a number of genes that have a CRE site in their promoter regions.

Overexpression of a dominant negative CREB transgene induces apoptosis in T cells following growth factor stimulation (16). Transgenic mice overexpressing a dominant negative CREB in the heart developed dilated cardiomyopathy (17). These studies suggest that CREB may contribute to cell survival and development. However, the precise role of CREB in the differentiation and proliferation of VSMC is not completely understood.

Previously, we have reported that CRE site is crucial for AngII-induced interleukin-6 expression in VSMC (18). However, the signaling pathway of AT1-R that regulates CREB activation remained unknown. In this report, we examined the signaling pathway of AT1-R responsible for CREB phosphorylation and the role of CREB in VSMC.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents-- Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum were purchased from Invitrogen. AngII was purchased from Peptide Institute. PD98059, KN93, and wortmannin were obtained from Research Biochemicals International. SB203580 is a generous gift from SmithKline Beecham Pharmaceuticals. H89 and W-7 were obtained from Biomol Research Laboratories Inc. AG1478 was obtained from Sigma. CV11974 was obtained from Takeda Chemical Industries Ltd., and PD123319 was obtained from Warner-Lambert, Park Davis Co. All antibodies used in the experiments were obtained from New England Biolabs Inc. except for the horseradish peroxidase-conjugated second antibodies (anti-rabbit or anti-mouse IgG, Vector Laboratories Inc.). [3H]leucine, [3H]thymidine, and [32P]dCTP were obtained from PerkinElmer Life Sciences. Unless mentioned otherwise, other chemical reagents were purchased from Wako Pure Chemicals.

Cell Culture-- VSMC were isolated from the thoracic aorta of Sprague-Dawley rats and maintained as described previously (18). Passages between 5 and 15 were used. VSMC were grown to confluent, growth-arrested in DMEM with 0.1% bovine serum albumin for 2 days, and used for the experiments.

Transfection of CRE-Luciferase Fusion DNA Construct to VSMC-- VSMC (3 × 105) were prepared in a 6-cm tissue culture dish. After 48 h, 5 µg of CRE-luciferase fusion DNA (three copies of the CRE site located upstream of the herpes simplex virus thymidine kinase promoter drive the luciferase gene (CLONTECH)) and 2 µg of the beta -galactosidase gene drive SV40 promoter-enhancer sequence were introduced to VSMC as described previously (18). After transfection, the cells were cultured in DMEM with 10% fetal bovine serum for 24 h, washed twice with phosphate-buffered saline, and stimulated with 10-7 mol/liter of AngII for 3 h in DMEM with 0.1% bovine serum albumin. The luciferase activity was measured and normalized by beta -galactosidase activity as described previously (18). Rat c-fos gene promoter (-436 bp-+45 bp) was cloned by polymerase chain reaction (19). The sequence was confirmed by dideoxy chain termination method in both sense and antisense strands, and the promoter region was subcloned into pGL3 basic luciferase expression vector (Promega).

Western Blot Analysis-- VSMC were lysed in a sample buffer (5 mmol/liter EDTA, 10 mmol/liter Tris-HCl, pH 7.6, 1% Triton X-100, 50 mmol/liter NaCl, 30 mmol/liter sodium phosphate, 50 mmol/liter NaF, 1% aprotinin, 0.5% pepstatin A, 2 mmol/liter phenylmethylsulfonyl fluoride, 5 mmol/liter leupeptin). Proteins were electrophoresed in 12% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Immobilon-P, MILLIPORE). The blots were blocked with TBS-T (20 mmol/liter Tris-HCl, pH 7.6, 137 mmol/liter NaCl, 0.1% Tween 20) containing 10% non-fat dry milk at room temperature for 1 h. Phosphorylated CREB at Ser-133 was detected by a phospho-CREB antibody (recognizes only the phosphorylated form) and ECL chemiluminescence (Amersham Biosciences) according to the manufacturer's instructions. The membranes were exposed to x-ray film. The membranes were stripped by incubating in a buffer containing 100 mmol/liter Tris-HCl, 2% SDS, and 100 mmol/liter 2-mercaptoethanol at 70 °C for 1 h and reprobed with an antibody against CREB (recognizes both the phosphorylated and nonphosphorylated forms) by the same procedure. The intensity of the bands was quantified by a MacBAS bioimaging analyzer (Fujifilm Co). Western blot analyses of ERK and p38MAPK were performed by the same procedure as that of CREB.

16-week-old Sprague-Dawley rats were anesthetized with an injection of sodium pentobarbital and killed by exsanguination, and the aorta was removed. After the adventitia was removed, the aorta was placed in a culture dish and stimulated with AngII in DMEM with 0.1% of bovine serum albumin for 1 h in the presence or absence of an isoform-specific antagonist for AngII receptor. Then, the lysate of aorta was prepared by incubating in the lysis buffer for 3 h and subjected to Western blot analysis as described above.

Expression of Dominant Negative Form of CREB-- A recombinant adenovirus vector expressing a mutant of CREB (Ad-CREB-M1) in which the phosphorylation site at Ser-133 was changed to alanine was a gift from Dr. Anthony J. Zeleznik (University of Pittsburgh) (20). VSMC grown to confluent were washed with PBS three times. Then, the cells were incubated with Ad-CREB-M1 or adenovirus vector expressing LacZ (Ad-LacZ) under gentle agitation for 2 h at room temperature. After infection, the cells were washed three times, cultured in DMEM with 0.1% bovine serum albumin for 2 days, and used for the experiments. Multiplicity of infection (m.o.i.) indicates the number of virus per cell added to culture dish.

Northern Blot Analysis-- Total RNA was prepared according to an acid guanidinium thiocyanate-phenol-chloroform extraction method (21), and Northern blot analysis of c-fos and 18 S ribosomal RNA was performed by a conventional method as described previously (18).

Measurement of Protein and DNA Synthesis-- VSMC were incubated with AngII (10-6 mol/liter), platelet-derived growth factor (PDGF)-BB (50 ng/ml), or serum (5%) for 24 h. The cells were labeled with [3H]leucine or [3H]thymidine (PerkinElmer Life Sciences) during the last 4 h. After labeling, the cells were washed with PBS, fixed in 10% trichloroacetic acid, and then washed with a mixture of ethanol and ether (2:1). The cells were lysed in 0.5 N NaOH, and incorporated [3H]leucine or [3H]thymidine was measured by a liquid scintillation counter.

Statistical Analysis-- Statistical analyses were performed by one-way analysis of variance and multiple comparison (Fisher) tests if appropriate. A p value less than 0.05 was considered significant. Data were expressed as mean ± S.E.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Activation of CRE-dependent Transcription by AngII-- We tested whether AngII activated CRE-dependent gene transcription by using CRE-luciferase reporter construct. As shown Fig. 1, normalized luciferase activity after AngII stimulation (10-7 mol/liter) was increased by 1.7-fold as compared with that of control (mean ± S.E., n = 6, p < 0.01). The induction of luciferase activity by AngII was blocked by an AT1-R antagonist, CV11974, but not by an AT2-R antagonist PD123319. This result suggests that AT1-R is responsible for the induction. Expression of the dominant negative form of CREB by Ad-CREB-M1 inhibited the up-regulation of CRE-luciferase activity by AngII.


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  		Fig. 1.   Activation of CRE-dependent transcription by AngII. CRE-luciferase fusion DNA (5 µg) was introduced to VSMC. At 24 h after transfection, the VSMC were incubated with or without AngII (10-7 mol/liter) for 3 h. The assay was also performed in the presence of CV11974 (10-5 mol/liter), an AT1-R antagonist, PD123319 (10-5 mol/liter), an AT2-R antagonist, or VSMC infected with Ad-CREB-M1 (A). The promoter region of the c-fos gene was fused to the luciferase gene (pGL3 basic) and introduced to VSMC, and then dominant negative CREB was overexpressed by Ad-CREB-M1 (B). The luciferase activity was standardized by the beta -galactosidase activity expressed by the cotransfected lacZ gene expression plasmid (2 µg). The normalized luciferase activity in VSMC without AngII stimulation was designated as 1.0. Results are expressed as mean ± S.E. (n = 6). **, p < 0.01 versus control.

CRE is one of the important cis-DNA elements regulating c-fos gene expression in response to mitogen (22). AngII increased c-fos promoter activity by 2-fold, which was abolished by overexpression of the dominant negative form of CREB (Fig. 1B). These results suggest that CRE and CREB play a critical role for AngII-induced gene expression in VSMC.

Phosphorylation of CREB at Ser-133 by AngII through AT1-R-- To investigate whether CREB is phosphorylated in response to AngII, we performed Western blot analysis using an antibody that only recognizes CREB species phosphorylated at Ser-133 (phospho-CREB antibody). AngII stimulated phosphorylation of CREB (Fig. 2A, upper panel) with a peak at 5 min of stimulation. AngII dose dependently induced phosphorylation of CREB at 5 min of stimulation (Fig. 2B, upper panel). Next, we determined the AngII receptor isoform responsible for CREB phosphorylation. Preincubation with CV11974 (10-5 mol/liter) completely abolished the CREB phosphorylation induced by AngII; however, PD123319 (10-5 mol/liter) did not show any effect (Fig. 2B). These data suggest that AT1-R may mediate the phosphorylation of CREB. The total level of CREB protein expression as detected by Western blot analysis with an antibody against CREB was unchanged after AngII stimulation (Fig, 2, A and B, lower panels). The ratio of phosphorylated CREB to total CREB measured by an imaging analyzer is shown in the right panel.


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  		Fig. 2.   Phosphorylation of CREB by AngII. Left panel, A, VSMC stimulated with AngII (10-7 mol/liter) for the indicated periods. B, VSMC stimulated for 5 min with various concentrations of AngII (from 10-10 to 10-6 mol/liter) or stimulated with AngII (10-7 mol/liter) for 5 min after 30 min of preincubation with CV11974 (10-5 mol/liter) or PD123319 (10-5 mol/liter). C, rat aorta stimulated ex vivo with AngII (10-7 mol/liter) in the presence of CV11974 (10-5 mol/liter) or PD123319 (10-5 mol/liter) for 1 h. Phosphorylation of CREB was detected by Western blot analysis using a phospho-specific CREB antibody (upper panel). The membrane was stripped and reprobed with a CREB antibody (lower panel). Right panel, the density of the specific band scanned and quantified by an imaging analyzer. The ratio of phosphorylated CREB to total CREB is shown. The ratio of untreated cells was designated as 1. Results are expressed as mean ± S.E. (n = 4). *, p < 0.05; **, p < 0.01 versus control.

To confirm that AngII stimulates CREB phosphorylation in intact aorta, rat aorta was stimulated with AngII ex vivo. Although it took about 1 h to detect, AngII induced phosphorylation of CREB in an AT1-R-dependent manner in intact aorta (Fig. 2C). The origin of CREB and phosphorylated CREB in intact aorta is not clear at this point. We observed, however, that AngII induced CREB phosphorylation in cultured aortic endothelial cells (data not shown), suggesting that both endothelial cells and VSMC are the source for CREB.

Multiple Protein Kinase Pathways Mediate AngII-induced CREB Phosphorylation-- A variety of protein kinases are reported to phosphorylate CREB. We investigated whether MAPK pathways were involved in AngII-induced CREB phosphorylation. AngII-induced CREB phosphorylation was partially blocked by PD98059 (30 µmol/liter), an ERK kinase (MEK) inhibitor, or SB203580 (10 µmol/liter), a p38MAPK inhibitor. A combination of PD98059 and SB203580 additionally inhibited the AngII-induced CREB phosphorylation (Fig. 3, A and B). The same concentration of PD98059 or SB203580 completely blocked the AngII-induced ERK phosphorylation and p38MAPK activation (23), respectively. These data suggest that these inhibitors sufficiently inhibited these MAPK pathways (Fig. 3C).


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  		Fig. 3.   Effects of MAPK inhibitors on AngII-mediated CREB phosphorylation. VSMC were preincubated with PD98059 (30 µmol/liter) and/or SB203580 (10 µmol/liter) for 30 min and stimulated with AngII (10-7 mol/liter) for 5 min. Western blot analysis was performed and analyzed as described in the legend for Fig. 2 (A). Results are expressed as mean ± S.E. (n = 4). *, p < 0.05; **, p < 0.01 versus AngII (AII). VSMC were preincubated with PD98059 (30 µmol/liter) or SB203580 (10 µmol/liter) for 30 min and stimulated with AngII (10-7 mol/liter) for 5 min (B). Western blot analyses using antibodies against phosphorylated forms of ERK (phospho-ERK) or ERK (left) and phosphorylated forms of p38MAPK (phospho-p38MAPK) and p38MAPK (right) were performed (C). Activation of ERK or p38MAPK by AngII (10-7 mol/liter for 5 min) was examined in the presence of CV11974 (10-5 mol/liter) or PD123319 (10-5 mol/liter). The same results were obtained in other independent experiments (n = 3), and a representative autoradiograph is shown.

W-7 and KN-93 are known as inhibitors of CaM and CaM-dependent protein kinase II, respectively (12, 24). As shown in Fig. 4, W-7 (50 µmol/liter) inhibited AngII-induced CREB phosphorylation. However, KN-93 had no significant effect. Inhibition of CaM by W-7 blocked the AngII-induced ERK activation (Fig. 4C) as reported previously (25), suggesting that the effect of W7 may be ascribed to inhibition of the ERK pathway rather than inhibition of CaMK. W7 and KN-93 completely blocked the A23187-induced CREB phosphorylation (Fig. 4D), indicating that the doses of these inhibitors sufficiently inhibited the calmodulin/CaMK pathway.


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  		Fig. 4.   Effects of inhibitors for CaM and CaM kinase on AngII-mediated CREB phosphorylation. VSMC were preincubated with W-7 (50 µmol/liter) or KN93 (10 µmol/liter) for 30 min and stimulated with AngII (10-7 mol/liter) for 5 min. Western blot analysis (A) and the densitometric analysis (B) were performed as described in the legend for Fig. 2. Results are expressed as mean ± S.E. (n = 4). **, p < 0.01 versus AngII (AII). N.S., not significant. VSMC were preincubated with W-7 (50 µmol/liter) for 30 min and stimulated with AngII (10-7 mol/liter) for 5 min (C). Western blot analysis of phosphorylated ERK (upper panel) and ERK (lower panel) were performed. The same results were obtained in other independent experiments (n = 3), and a representative autoradiograph is shown. VSMC were preincubated with W-7 (50 µmol/liter) or KN93 (10 µmol/liter) for 30 min and stimulated with A23187 (10 µmol/liter) for 5 min (D). Western blot analysis was performed as described in the legend for Fig. 2. The same results were obtained in other independent experiments (n = 3), and a representative autoradiograph is shown.

A recent study suggests that CREB is a downstream target of Akt/protein kinase B that is activated by PI3K (15). Preincubation with wortmannin (50 nmol/liter, 30 min) that inhibits PI3K did not affect AngII-induced CREB phosphorylation (Fig. 5, A and B). However, wortmannin inhibited insulin-induced CREB phosphorylation (Fig. 5C), indicating that the PI3K-Akt pathway was sufficiently inhibited by this concentration of wortmannin in our VSMC.


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  		Fig. 5.   Effect of a PI3K inhibitor on AngII-mediated CREB phosphorylation. VSMC were preincubated with wortmannin (50 nmol/liter) for 30 min and stimulated with AngII (10-7 mol/liter) for 5 min. Western blot analysis (A) and the densitometric analysis (B) were performed as described in the legend for Fig. 2. Results are expressed as mean ± S.E. (n = 4). VSMC were preincubated with wortmannin (50 nmol/liter) for 30 min and stimulated with insulin (10-7 mol/liter) for 10 min (C). Western blot analysis was performed as described in the legend for Fig. 2. The same results were obtained in other independent experiments (n = 3), and a representative autoradiograph is shown. N.S., not significant.

Next, we examined the effect of H89, a specific PKA inhibitor, on AngII-induced CREB phosphorylation. Preincubation with H89 partially inhibited AngII-induced CREB phosphorylation (Fig. 6, A and B). H89 at this concentration completely inhibited the forskolin-induced CREB phosphorylation (Fig. 6C) but did not affect AngII-induced ERK or p38MAPK activation (Fig. 6D).


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  		Fig. 6.   Effect of a PKA inhibitor on AngII-mediated CREB phosphorylation. VSMC were preincubated with H89 (10 µmol/liter) for 30 min and then stimulated with AngII (10-7 mol/liter) for 5 min. Western blot analysis (A) and the densitometric analysis (B) were performed as described in the legend for Fig. 2. Results are expressed as mean ± S.E. (n = 4). **, p < 0.01 versus AngII (AII). VSMC were preincubated with H89 (10 µmol/liter) for 30 min and stimulated with forskolin (10 µmol/liter) for 5 min (C). Western blot analysis was performed as described in the legend for Fig. 2. The same results were obtained in other independent experiments (n = 3), and a representative autoradiograph is shown. The effect of H89 on AngII-induced ERK and p38MAPK activation was examined as described in the legend for Fig. 3 (D). The same results were obtained in other independent experiments (n = 3), and a representative autoradiograph is shown.

Transactivation of Epidermal Growth Factor-Receptor (EGF-R) Is Critical for AngII-induced CREB Phosphorylation-- Recent reports have shown that transactivation of EGF-R is indispensable for the signaling of certain G-protein-coupled receptors (26) including AT1-R (25). We, therefore, examined the effect of AG1478, a specific EGF-R inhibitor, on AngII-induced CREB phosphorylation. As shown in Fig. 7, A and B, AG1478 completely abolished AngII-induced CREB phosphorylation. AG1478 also inhibited AngII-induced activation of ERK (Fig. 7C) and p38MAPK (Fig. 7D). However, inhibition of the protein kinase C pathway by prolonged exposure to phorbol myristate acetate (PMA) or GF109203X did not affect the activation of CREB, ERK, or p38MAPK by AngII (Fig. 8, A-D). Prolonged exposure to PMA or GF109203X completely inhibited PMA-induced CREB phosphorylation (Fig. 8E), suggesting that these treatments sufficiently suppressed PKC pathway.


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  		Fig. 7.   Effect of AG1478 on AngII-induced CREB phosphorylation. VSMC were incubated with AG1478 (2.5 µmol/liter) for 30 min and then stimulated with AngII (10-7 mol/liter) for 5 min. Western blot analysis (A) and the densitometric analysis (B) were performed as described in legend for Fig. 2. Results are expressed as mean ± S.E. (n = 4). **, p < 0.01 versus AngII (AII). The effect of AG1478 on AngII-induced ERK (C) and p38MAPK (D) activation was examined as described in the legend for Fig. 3. The same results were obtained in other independent experiments (n = 3), and a representative autoradiograph is shown.


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  		Fig. 8.   Effect of PKC inhibitors on AngII-induced CREB phosphorylation. VSMC were incubated with PMA (1 µmol/liter) for 24 h or GF109203X (1 µmol/liter) for 30 min and then stimulated with AngII (10-7 mol/liter) for 5 min. Western blot analysis (A) and the densitometric analysis (B) were performed as described in the legend for Fig. 2. Results are expressed as mean ± S.E. (n = 4). **, p < 0.01 versus control (con). O/N, overnight exposure. The effect of prolonged exposure to PMA or GF109203X on AngII-induced ERK (C) and p38MAPK (D) activation was examined as described in the legend for Fig. 3. The same results were obtained in other independent experiments (n = 3), and a representative autoradiograph is shown. The effect of prolonged exposure to PMA or GF109203X on PMA (1 µmol/liter for 10 min)-induced CREB activation was examined as described in the legend for Fig. 2 (E). The same results were obtained in other independent experiments (n = 3), and a representative autoradiograph is shown.

Effect of Overexpression of Dominant Negative Form of CREB on AngII-induced c-fos Expression and Protein Synthesis in VSMC-- To clarify the role of CREB in AngII signaling, we overexpressed the dominant negative form of CREB by an adenovirus vector. The immunoreactivity of CREB was increased in an m.o.i.-dependent manner (Fig. 9A, lower panel). Phosphorylation of CREB by AngII was attenuated by the infection of Ad-CREB-M1 (p < 0.01). We used overexpression of beta -galactosidase by Ad-LacZ as a negative control for the infection of adenovirus. As shown in Fig. 9B, the infection of Ad-LacZ did not affect AngII-induced CREB phosphorylation. Ad-CREB-M1 but not Ad-LacZ suppressed AngII-induced c-fos mRNA expression (Fig. 9C).


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  		Fig. 9.   Suppression of AngII-induced leucine incorporation and c-fos mRNA expression by Ad-CREB-M1. VSMC were infected with (A) 10 or 30 m.o.i. of Ad-CREB-M1 that express unphosphorylatable CREB or (B) 30 m.o.i. of Ad-LacZ and stimulated with AngII (10-7mol/liter) for 5 min. Phosphorylation of CREB and total CREB expression levels were detected by Western blot analysis as described in the legend for Fig. 2. *, p < 0.01 versus without Ad-CREB-M1. (n = 3) #, p < 0.01 versus AngII without Ad-CREB-M1. VSMC were infected with Ad-CREB-M1 (30 m.o.i.) or Ad-LacZ (30 m.o.i.) and then stimulated with AngII (10-7mol/liter) for 30 min (C). Total RNA was prepared and examined by Northern blot analysis. *, p < 0.01 versus without Ad-CREB-M1 (n = 3). A representative autoradiograph is shown.

Next, we examined the effect of Ad-CREB-M1 on AngII-induced protein and DNA synthesis. The infection of Ad-CREB-M1 suppressed AngII-induced leucine incorporation (Fig. 10A), whereas Ad-LacZ did not affect the AngII-induced leucine incorporation. We also measured AngII-induced [3H]thymidine incorporation. However, AngII caused a very small increase in thymidine incorporation in our VSMC as reported previously (27), and we failed to see a significant effect of Ad-CREB-M1 on AngII-induced [3H]thymidine incorporation (Fig. 10B). Ad-CREB-M1 did not affect PDGF-BB- or serum-induced incorporation of leucine (Fig. 10C), suggesting that the infection of Ad-CREB-M1 is not toxic.


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  		Fig. 10.   Effect of Ad-CREB-M1 on AngII-induced protein and DNA synthesis. A and B, VSMC were infected with Ad-CREB-M1 (30 m.o.i.) or Ad-LacZ (30 m.o.i.) and then incubated with AngII (10-7mol/liter) for 24 h. VSMC were infected with Ad-CREB-M1 (30 m.o.i.) and then incubated with PDGF-BB (50 ng/ml) or serum (5%) for 24 h (C). Incorporation of [3H]leucine (A and C) or [3H]thymidine (B) was measured (n = 4) and normalized with the cell number. Results are expressed as percent of control culture. Data are shown as mean ± S.E. *, p < 0.05 VS control. NS, not significant.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This is, to our knowledge, the first report showing AT1-R-regulated CREB phosphorylation and CRE-dependent gene transcription. AngII activated several protein kinases that phosphorylated CREB at Ser-133 through AT1-R. Our results suggest that AngII-induced CREB phosphorylation is mediated by at least three distinct pathways: (i) a MEK-ERK pathway that can be blocked by PD98059, (ii) a p38MAPK pathway that can be blocked by SB203580, and (iii) a PKA-dependent pathway that can be blocked by H89. In addition, transactivation of EGF-R is critical for CREB phosphorylation.

ERK plays an important role for the signaling of many growth factors. AngII activates ERK through transactivation of EGF-R (25). Recently, nerve growth factor (NGF) (13) and EGF (28) were reported to phosphorylate and activate CREB through the ERK-p90rsk2-dependent pathway. AngII-induced p90rsk2 activation is also ERK-dependent (29), suggesting that this pathway may be important for AngII-induced CREB phosphorylation. In contrast to EGF or NGF, fibroblast growth factor (FGF)-induced CREB phosphorylation is mediated by MAPKAP kinase-2 (14) that lies immediately downstream from p38MAPK. AngII was also reported to activate p38MAPK (6). Recently, a novel protein kinase that is activated by both ERK and p38MAPK was reported and designated mitogen- and stress-activated protein kinase-1 (MSK-1) (30). MSK-1 phosphorylates CREB in response to FGF or NGF in PC12 cells. At present, it is not clear which kinase is directly responsible for AngII-induced CREB phosphorylation downstream from p38MAPK or ERK.

Transactivation of EGF-R is a critical signaling step for certain G-protein-coupled receptors such as endothelin, thrombin, and AngII receptor (25, 26). We showed that AngII activates ERK and p38MAPK in an EGF-R-dependent manner, suggesting that inhibition of AngII-induced CREB phosphorylation by AG1478 may be ascribed to the suppression of these MAPK pathways.

An increase in intracellular Ca2+ and activation of the calcium/CaM pathway is crucial for the AngII signaling (25). However, the downstream CaM-dependent kinase activated by AngII is not clear. Recently, Abraham et al. (31) reported that inhibition of CaM kinase II by KN-93 partially inhibited AngII-induced ERK phosphorylation. A previous study showed that CaM kinase II and IV were able to phosphorylate CREB (12). Our study showed that KN-93 did not affect AngII-induced CREB phosphorylation, whereas an inhibition of CaM by W-7 partially suppressed it. These data may suggest that CaM kinase IV rather than CaM kinase II is responsible for AngII-induced CREB phosphorylation. However, W-7 completely suppressed the AngII-induced ERK activation as reported previously (25), suggesting that the effect of W-7 may be ascribed to inhibition of the ERK pathway rather than inhibition of the CaM kinase IV pathway. Furthermore, it was also reported that CaM kinase IV was not expressed in VSMC (32). Therefore it is unlikely that CaM kinase IV phosphorylates CREB in response to AngII.

To our surprise, AngII-induced CREB phosphorylation was partially inhibited by H89, a PKA inhibitor. The majority of reports showed that AngII inhibits adenylate cyclase (33, 34). However, the role of adenylate cyclase in AngII signaling is still enigmatic because there are several studies that have reported activation of adenylate cyclase in response to AngII (35). Further investigation is necessary to determine whether cAMP production and PKA activity are up-regulated or down-regulated by AngII in VSMC. We showed that H89 inhibited the forskolin-induced CREB phosphorylation but did not affect the AngII-induced ERK or p38MAPK activation, suggesting that H89 inhibited AngII-induced CREB phosphorylation independently of MAPK pathways. Recently, Impey et al. (36) reported an obligatory role of PKA for the nuclear translocation of ERK and subsequent CREB activation in response to NGF in neuronal culture. They failed to detect an elevation of intracellular cAMP level by NGF and proposed that basal PKA activity was critical for the nuclear translocation of ERK. Therefore it may be possible that basal PKA activity rather than activation of PKA is necessary for AngII-induced CREB activation.

Although AngII induced CREB phosphorylation by severalfold, AngII increased CRE promoter activity by ~2.0-fold. The reason for this difference is not clear. However, Brindle et al. (37) and Ginty et al. (38) reported that the ability to activate CRE-dependent gene transcription is different among signaling pathways despite the similar level of CREB phosphorylation. A recent report by Mayr et al. (39) may explain this differential effect on CREB phosphorylation and CRE-dependent gene transcription. They showed that the CREB-CBP complex induced by mitogenic signals such as NGF or EGF is less stable than that induced by cAMP in the nucleus. Therefore the relative instability of AngII-induced CREB-CBP complex may account for the weak activation of CRE-dependent gene transcription by AngII. Alternatively, basal promoter activity of these luciferase constructs is relatively high in our VSMC, and the up-regulation of luciferase activity by AngII may not be prominent.

Ad-CREB-M1 almost completely suppressed AngII-induced leucine incorporation. Because a number of genes are reported to have a CRE site in the promoter region, it is difficult to determine the CREB-dependent gene(s) that is responsible for AngII-induced leucine incorporation in VSMC. There may be a few CREB-dependent genes that are important for VSMC hypertrophy. Alternatively, partial suppression of gene expression, such as the effect of Ad-CREB-M1 on AngII-induced c-fos expression that we observed, might accumulate when CREB is inhibited and the cumulative effects eventually result in inhibition of leucine incorporation. Further study is necessary to identify the target gene(s) that is inhibited by the dominant negative form of CREB. At any rate, AD-CREB-M1 is not toxic because it did not affect PDGF-BB- or serum-induced leucine incorporation.

Ad-CREB-M1 attenuated AngII-induced CREB phosphorylation, which may contribute to the inhibition of CREB function. However, the mechanism by which CREB-M1 inhibits CREB function is believed to replace the endogenous CREB with the mutated CREB rather than to inhibit phosphorylation of endogenous CREB (20). Because CREB can dimerize with the ATF-1 transcription factor, it is possible that the effect of CREB-M1 may be ascribed to the sequestration of ATF-1. This possibility cannot be excluded at this point.

VSMC express alpha 1 and beta -adrenergic receptors. A recent study has shown that stimulation of alpha 1 receptor induces CREB phosphorylation (40). This study and our data suggest that signaling of the adrenergic system and renin angiotensin system may converge on CRE-dependent gene transcription and the possible involvement of CREB in vascular remodeling. Although the precise role of CREB in VSMC is not clarified, CREB activation pathway may represent a potentially novel target for the treatment of atherosclerosis.

A growing body of evidences suggests that the renin angiotensin system plays a critical role in the processes of cardiac hypertrophy, heart failure, and atherosclerosis. Our results suggest that the pathophysiological relevance for activation of AT1-R may involve transcriptional activation of the gene containing the CRE enhancer element.

    FOOTNOTES

* This work was supported in part by a grant-in-aid for Scientific Research on Priority Areas (C) "Medical Genome Science" from the Ministry of Education, Culture, Sports, Science and Technology of Japan and Kobayashi Magobe Memorial Medical Foundation, Okayama, Japan (to T. I.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, 812-8582 Fukuoka, Japan. Tel.: 81-92-642-5361; Fax 81-92-642-5374; E-mail: ichiki@cardiol.med. kyushu-u.ac.jp.

Published, JBC Papers in Press, March 20, 2002, DOI 10.1074/jbc.M110430200

    ABBREVIATIONS

The abbreviations used are: AngII, angiotensin II; CRE, cyclic AMP-response element; CREB, CRE-binding protein; CBP, CREB-binding protein; VSMC, vascular smooth muscle cells; AT1-R, AngII type 1 receptor; AT2-R, AngII type 2 receptor; Ad, adenovirus; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, Jun N-terminal kinase; MAPKAP, MAPK-activated protein; MEK, MAPK/ERK kinase; PKA, protein kinase A; PI3K, phosphatidylinositol 3-kinase; EGF, epidermal growth factor; EGF-R, EGF receptor; PDGF-BB, platelet-derived growth factor-BB; NGF, nerve growth factor; FGF, fibroblast growth factor; CaM, calmodulin; CaMK, CaM kinase; DMEM, Dulbecco's modified Eagle's medium; PMA, phorbol myristate acetate; m.o.i., multiplicity of infection; ATF, activating transcription factor.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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