cAMP-Response Element-Binding Protein Mediates Tumor Necrosis Factor-α–Induced Vascular Smooth Muscle Cell Migration
Objective— Migration of vascular smooth muscle cells (VSMCs) contributes to formation of vascular stenotic lesions such as atherosclerosis and restenosis after angioplasty. Previous studies have demonstrated that tumor necrosis factor-α (TNF-α) is a potent migration factor for VSMCs. cAMP-response element-binding protein (CREB) is the stimulus-induced transcription factor and activates transcription of target genes such as c-fos and interleukin-6. We examined whether CREB is involved in TNF-α–induced VSMC migration.
Methods and Results— TNF-α induced CREB phosphorylation with a peak at 15 minutes of stimulation. Pharmacological inhibition of p38 mitogen-activated protein kinase (p38-MAPK) inhibited TNF-α–induced CREB phosphorylation. Adenovirus-mediated overexpression of dominant-negative form of CREB suppressed TNF-α–induced CREB phosphorylation and c-fos mRNA expression. VSMC migration was evaluated using a Boyden chamber. Overexpression of dominant-negative form of CREB suppressed VSMC migration as well as Rac1 expression induced by TNF-α. Overexpression of dominant-negative Rac1 also inhibited TNF-α–induced VSMC migration.
Conclusion— Our results suggest that p38-MAPK/CREB/Rac1 pathway plays a critical role in TNF-α–induced VSMC migration and may be a novel therapeutic target for vascular stenotic lesion.
Accumulation of vascular smooth muscle cells (VSMCs) in the intima of arteries is one of the most prominent cellular features of the atherosclerotic plaque and of the neointimal hyperplasia that causes restenosis after angioplasty.1,2 VSMC accumulation in the intima results from cell proliferation and directed migration of VSMCs from the media. These processes are regulated by various factors that are produced locally at the sites of vascular lesion by macrophages, T-lymphocytes, platelets, endothelial cells, and VSMCs.
Tumor necrosis factor-α (TNF-α) is a potent proinflammatory cytokine produced by activated macrophages, monocytes, and lymphocytes. TNF-α is expressed actively in atherosclerotic lesions and in the intima of arteries after injury.3 In vivo experiments demonstrated a critical role of TNF-α in the neointimal formation of vascular stenosis.4,5 TNF-α blockade by soluble TNF-α receptor suppressed coronary artery neointimal formation after cardiac transplantation in rabbits.4 Although previous study demonstrated that TNF-α is a potent migration factor for VSMCs,6 its signaling pathway involved in VSMC migration is not fully elucidated.
cAMP-response element (CRE)-binding protein (CREB) is a 43-kDa nuclear transcription factor belonging to the CREB/activating transcription factor (ATF) family.7,8 Phosphorylation of serine residue at 133 (Ser133) is necessary for transcriptional activation. Ser133 phosphorylation is mediated by a variety of protein kinase pathways such as (1) protein kinase A (PKA), (2) Ca2+/calmodulin-dependent protein kinase (CaMK) II,9 (3) extracellular signal-regulated kinase (ERK),10 (4) p38 mitogen-activated protein kinase (p38-MAPK),11 and (5) phosphatidylinositol 3-kinase (PI3K).12 Many reports have defined a role of CREB in proliferation and differentiation of numerous tissues; however, a role of CREB in cell migration remains largely unknown.
In this report, we showed that TNF-α activated CREB through p38-MAPK and that overexpression of dominant-negative form of CREB inhibited TNF-α–induced VSMC migration.
Materials and Methods
DMEM was purchased from GIBCO/BRL. FBS was purchased from BioWhittaker. BSA, ionomycin, and KN-93 were purchased from Sigma. Recombinant human TNF-α was a generous gift from Dainippon Pharmaceutical Co. (Osaka, Japan). PD98059 and wortmannin were purchased from Biomol. SB203580 was a gift from GlaxoSmithKline (Research Triangle Park, NC). H89 was purchased from Seikagaku Kogyo. Rac1 and RhoA antibodies were purchased from Santa Cruz Biotechnology. Horseradish peroxidase–conjugated second antibodies (anti-rabbit or anti-mouse IgG) were purchased from Vector Laboratories. Other antibodies used in the experiments were obtained from Cell Signaling Technology. Other chemical reagents were purchased from Wako Pure Chemical unless mentioned specifically.
Human coronary artery VSMCs were purchased from BioWhittaker. Passages between 7 and 12 were used for the experiments. VSMCs were grown to confluence, growth-arrested in DMEM with 0.1% BSA for 2 days, and used for the experiments.
Western Blot Analysis
VSMCs were lysed in a sample buffer (5 mmol/L EDTA, 10 mmol/L Tris-HCl, pH 7.6, 1% Triton X-100, 50 mmol/L NaCl, 30 mmol/L sodium phosphate, 50 mmol/L NaF, 1% aprotinin, 0.5% pepstatin A, 2 mmol/L phenylmethylsulfonyl fluoride, and 5 mmol/L leupeptin). Western blot analyses of CREB, ERK, Akt, Rac1, and RhoA were performed as described previously.13
Adenovirus Vector Expressing LacZ and Dominant-Negative Form of CREB
A recombinant adenovirus vector expressing a mutant of CREB (AdCREB M1)14 in which the phosphorylation site at Ser133 was changed to alanine was a generous gift from Dr Anthony J. Zeleznik (University of Pittsburgh, Pennsylvania). A recombinant adenovirus vector expressing Asn17-Rac1 (AdDNRac1) was generated in our laboratory by Adenovirus Expression Vector Kit (Takara) according to instructions of the manufacturer. Confluent VSMCs were washed 2× with PBS and incubated with AdCREB M1, AdDNRac1, or adenovirus vector expressing LacZ (AdLacZ) under gentle agitation for 2 hours at room temperature. Then the cells were washed 3×, cultured in DMEM with 0.1% BSA for 2 days, and used for the experiments. Multiplicity of infection (moi) 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. Northern blot analysis of c-fos, Cdc42, and 18S rRNA was performed as described previously.13 Radioactivity of hybridized bands of c-fos mRNA, Cdc42 mRNA, and 18S rRNA was quantified with a MacBAS Bioimage Analyzer (Fuji).
Cell migration was analyzed in a Boyden chamber housing a collagen I-precoated polycarbonate membrane with 8.0-μm pores (Chemicon). Lower chambers were filled with or without TNF-α (10 ng/mL) in serum-free medium. Then 4.0×104 cells were placed on the upper side of the membrane and allowed to migrate through the pores. After 4 hours of incubation, the number of cells that migrated to the lower surface of the membrane was counted per ×200 high-power fields (HPFs). The cell number of 10 randomly chosen HPFs was counted per well.
Preparation of Nuclear Extracts and Gel Mobility Shift Assay
Preparation of nuclear extracts and gel mobility shift assay were performed as described previously.15 DNA probes of Rac-CRE (5′-GAACTCGTGACCTCAGGTGAT-3′: −521 bp to ≈ −540 bp from transcription initiation site) and nuclear factor κB (NF-κB; 5′-AGATGAGGGGACTTTCCCAGGC-3′, consensus sequence) were labeled with 32P. Ten micrograms of nuclear extracts were incubated with 1×105 counts per minute of labeled DNA probe for 30 minutes at room temperature and electrophoresed on 4% acrylamide gel. Fifty-fold molar excess of unlabeled DNA was added as a competitor. After electrophoresis, gels were dried and exposed to x-ray films.
Statistical analysis was performed with 1-way ANOVA and Fisher test if appropriate. P<0.05 was considered statistically significant. Data are shown as mean±SEM.
CREB Phosphorylation at Ser133 by TNF-α
To examine whether CREB is phosphorylated in response to TNF-α, we performed Western blot analysis using an antibody that only recognized phosphorylated form of CREB at Ser133. TNF-α stimulated CREB phosphorylation with a peak at 15 minutes of stimulation (Figure 1A). TNF-α dose-dependently increased CREB phosphorylation with a peak at 1 ng/mL after 15 minutes of stimulation (Figure 1B).
p38-MAPK Pathway Mediates TNF-α–Induced CREB Phosphorylation
Several protein kinases are reported to phosphorylate CREB.9–12 We examined which pathway was responsible for TNF-α–induced CREB phosphorylation. SB203580 (10 μmol/L), a p38-MAPK inhibitor, completely blocked TNF-α–induced CREB phosphorylation (Figure 2). TNF-α stimulated p38-MAPK phosphorylation with a peak at 15 minutes of stimulation (Figure 3A), and the same concentration of SB203580 blocked TNF-α–induced phosphorylation of p38-MAPK and ATF2, which is a downstream target molecule of p38-MAPK (Figure 3B). SB203580 did not affect TNF-α–induced c-Jun N-terminal kinase (JNK) or ERK phosphorylation (Figure 3B). PD98059 (10 μmol/L), an ERK kinase (MAPK kinase) inhibitor, wortmannin (50 nmol/L), an inhibitor of PI3K, KN-93 (10 μmol/L), an inhibitor of CAMKII, and H89 (1 μmol/L), an inhibitor of PKA, did not affect TNF-α–induced CREB phosphorylation (Figure 2). The same concentration of PD98059 blocked the TNF-α–induced ERK phosphorylation. Wortmannin blocked TNF-α–induced Akt phosphorylation. H89 and KN93 at the same concentration blocked forskolin and ionomycin-induced CREB phosphorylation, respectively (Figure I, available online at http://atvb.ahajournals.org.). These data confirmed that these protein kinase inhibitors at the concentration we used sufficiently inhibited target protein kinase pathways. Together, these data suggest that p38-MAPK pathway is important for TNF-α–induced CREB phosphorylation.
Dominant-Negative Form of CREB Overexpression Inhibits TNF-α–induced c-fos Expression
To clarify the role of CREB in TNF-α signaling, we overexpressed dominant-negative form of CREB by an adenovirus vector (AdCREB M1). We used AdLacZ as a negative control for adenovirus infection. CREB phosphorylation by TNF-α was attenuated by AdCREB M1 infection (Figure II, available online at http://atvb.ahajournals.org). CRE is one of the important cis-DNA elements regulating c-fos gene expression. AdCREB M1 suppressed TNF-α–induced c-fos mRNA expression (Figure II). Although AdLacZ infection slightly upregulated basal c-fos mRNA expression, the increase was statistically insignificant; and the response of c-fos gene expression to TNF-α was not affected. These data suggest the critical role of CREB in TNF-α–induced c-fos gene expression.
AdCREB M1 Inhibits TNF-α–Induced VSMC Migration
TNF-α is known to induce VSMC migration.6,16 To evaluate the role of CREB in regulation of TNF-α–induced VSMC migration, we performed cell migration assay using a Boyden chamber. AdCREB M1 infection suppressed TNF-α–induced VSMC migration; however, AdLacZ infection did not (Figure 4A). This suggests that CREB mediates TNF-α–induced migration. Next, we examined the effect of SB203580 on TNF-α–induced VSMC migration. VSMC treatment with SB203580 decreased VSMC migration induced by TNF-α (Figure 4B). These data suggest that p38-MAPK/CREB pathway is critical for TNF-α–induced VSMC migration. The quantity of VSMC migration in this study is almost the same as that reported previously.16 We reported previously that AdCREBM1 induced VSMC apoptosis after 4 days of infection.17 We examined cell migration after 2 days of AdCREB M1 infection. At this point, staining with Hoechst 33258 showed that AdCREB M1 infection increased apoptosis cells (1.6%) compared with LacZ infection (0.2%). However, AdCREB M1 infection decreased VSMC migration by 38% compared with AdLacZ infection in this study, therefore, the effect of AdCREB M1 on cell viability in the condition of migration assay may be negligible.
Platelet-derived growth factor (PDGF)-BB is one of the most potent chemoattractants for VSMCs.1,2 AdCREB M1 infection did not suppress PDGF-BB–induced VSMC migration (Figure 4C). This suggests that CREB may not be involved in PDGF-BB–induced migration.
AdCREB M1 Inhibits TNF-α–Induced Rac1 Expression
The Rho family of small guanosine triphosphatases (GTPases), such as RhoA and Rac1, is an essential regulator of actin cytoskeletons and cell migration.18,19 We used TFSEACH (transcription factor–binding sites) to search the promoter region of these small G-proteins. TFSEACH results showed the presence of possible CRE site in the promoter of RhoA, Rac1, and Cdc42. Therefore, we hypothesized that CREB regulated the Rho family GTPase expression. AdCREB M1 infection suppressed Rac1 protein expression induced by TNF-α; however, LacZ did not (Figure 5). TNF-α did not affect RhoA or Cdc42 expression (Figure 5). These data suggest that CREB regulates TNF-α–induced Rac1 protein expression in VSMCs. Several studies demonstrated that Rac1 inhibition attenuated cell migration,20–22 and we examined the role of Rac1 on TNF-α–induced VSMC migration. AdDNRac1 infection suppressed TNF-α–induced VSMC migration (Figure 4D). This suggests that one of the CREB target molecules involved in TNF-α–induced VSMC migration is Rac1. We performed an electrophoretic mobility shift assay to examine whether TNF-α increases CREB DNA binding to the CRE site in the Rac1 promoter. TNF-α increased NF-κB binding activity to the consensus NF-κB sequence with the peak at 1 hour of stimulation; however, it did not affect CREB binding activity to the CRE site in the Rac1 promoter (Figure III, available online at http://atvb.ahajournals.org). CREB binding was specific because the band was eliminated by 50 molar excess of unlabeled competitor, and the band was supersifted by addition of an antibody against CREB. Many studies indicate that CREB phosphorylation on Ser133 does not alter CREB/DNA interaction.23,24 Our data also indicated that CREB phosphorylation did not increase CREB binding to Rac1 gene promoter.
In the present study, we showed that CREB was activated by TNF-α and mediated TNF-α–induced VSMC migration. Activation of CREB by TNF-α and TNF-α–induced VSMC migration was dependent on p38-MAPK pathway. One of the target molecules of CREB involved in TNF-α–induced VSMC migration may be Rac1, a Rho family small GTPase.
Recent evidence suggests that MAP kinases play the major role in stress-induced cellular responses including cell proliferation, survival, and apoptosis.25 It was reported that ERK pathway mediated VSMC migration induced by TNF-α.16 In the present study, we showed that p38-MAPK inhibition by SB203580 reduced TNF-α–induced VSMC migration. This suggests that p38-MAPK also plays an important role in TNF-α–induced VSMC migration. Recently, several reports have shown that activation of the p38-MAPK pathway is important for cell migration and actin reorganization. In vascular endothelial cells, it was shown that p38-MAPK plays a critical role in oxidative stress–induced actin reorganization26 and PDGF-induced cell migration.27 p38-MAPK activation regulates PDGF-induced tracheal smooth muscle cell migration by signaling to the 27-kDa heat shock protein.28 Our results indicating that p38-MAPK pathway is critical for VSMC migration induced by TNF-α are consistent with these reports. In the downstream of p38-MAPK, MAPK-activated protein kinase-211 and mitogen- and stress-activated protein kinase-129 are activated. These MAPKs are believed to phosphorylate CREB. However, it is not clear at the moment which kinase is responsible for CREB phosphorylation induced by TNF-α.
Members of the Rho family of small GTPase are key regulators of actin cytoskeletal dynamics.18,19 Cdc42 activation induces actin polymerization at the cell front to form long and thin extensions (filopodia). Rac activation induces formation of broader web-like extensions (lamipodia). Rho induces further organization of actin into bundles and production of large, highly organized structures termed focal adhesions. The database search for CRE site in the promoter regions revealed the presence of possible CRE site in all Rho family of small GTPase. However, in the present study, we demonstrated that CREB inhibition reduced TNF-α–induced VSMC migration and Rac1 protein expression, and TNF-α did not affect expression of RhoA or Cdc42. Furthermore, adenovirus-mediated expression of a dominant-negative form of Rac suppressed TNF-α–induced VSMC migration. This suggests that Rac1 gene activation through CRE/CREB may be involved in VSMC migration induced by TNF-α. Accumulating evidence has demonstrated that Rac1 inhibition attenuated cell migration. In in vitro wound healing assay using rat embryo fibroblasts, microinjection of wound edge cells with dominant-negative Rac1 prevented lamellipodia formation and membrane ruffing, and there was no forward movement.20 In VSMCs and human umbilical vein endothelial cells, adenovirus-mediated expression of a dominant-negative form of Rac1 attenuated cell migration.21 Furthermore, Rac1 inactivation in the Drosophila ovary prevents migration of border cells during oogenesis from the anterior tip, through the nurse cells, and to the oocyte.22 Results of these studies are consistent with our results. Although Rac1 has been shown to associate with several effector molecules such as POR1 and PAK, WAVE family is thought to be the main downstream effector molecule of Rac1 for formation of lamellipodia and membrane ruffle.30 WAVE regulates membrane ruffle formation by activating the Arp2/3 complex.31 The effect of dominant-negative CREB on these proteins is not clear at the moment. In the present study, we used Boyden chamber to assess VSMC migration. However, it is not clear whether dominant-negative CREB affects other processes of cell function such as VSMC attachment to the membrane. It is difficult to separate these processes in our assay system. Therefore, we believe that the results of Boyden chamber assay reflect several steps of cell migration, including adhesion and movement.
Although it is generally accepted that phosphorylation enhances transactivation potential of CREB (its ability to recruit the transcriptional apparatus through its transactivation domain), there is conflicting evidence regarding whether phosphorylation also regulates the DNA-binding activity of CREB. In vitro binding studies indicate that CREB phosphorylation on Ser133 does not alter affinity of CREB for a palindromic CRE, although stimulatory effects of Ser133 phosphorylation on half site CRE binding have been observed.32 Genomic foot-printing has also revealed that elevated levels of cAMP increased CRE occupancy of the tyrosine aminotransferase in vivo.33 Several other studies have failed to detect phosphorylation-induced changes in the CREB/DNA interaction.23,24 It is unclear why different laboratories have reached such disparate conclusions. We showed that TNF-α induced CREB phosphorylation but did not affect DNA-binding activity of CREB to Rac1 promoter. These data indicate that CREB phosphorylation stimulates Rac1 transcription not through regulation of its DNA-binding activity.
Klemm et al reported that CREB activation decreased PDGF-BB–induced migration in bovine aortic VSMCs.34 However, many chemoattractants such as PDGF-BB,35 insulin-like growth factor I,36 and angiotensin II37 are known to stimulate CREB phosphorylation as well as proliferation, hypertrophy, and migration of VSMCs. These results and our data strongly suggest that CREB may be involved in these processes. The reason for the discrepant results between Klemm et al and ours is not clear at this point and requires further investigation.
Vascular remodeling in response to injury requires rearrangement in the VSMC cytoskeleton and includes migration of activated VSMCs from the tunica media into the intimal layer of the arterial wall. The data presented in this study suggest that p38-MAPK/CREB/Rac pathway may play a critical role in vascular stenotic disease and could be a therapeutic target for prevention of restenosis or atherosclerosis.
This study was supported in part by grants from Takeda Medical Research Foundation and Mitsubishi Pharma Research Foundation, and a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (14570673).
Consulting Editor for this article was Alan M. Fogelman, MD, Professor of Medicine and Executive Chair, Departments of Medicine and Cardiology, UCLA School of Medicine, Los Angeles, CA.
- Received February 27, 2004.
- Accepted June 25, 2004.
Clausell N, Molossi S, Sett S, Rabinovitch M. In vivo blockade of tumor necrosis factor-α in cholesterol-fed rabbits after cardiac transplant inhibits acute coronary artery neointimal formation. Circulation. 1994; 89: 2768–2779.
Rectenwald JE, Moldawer LL, Huber TS, Seeger JM, Ozaki CK. Direct evidence for cytokine involvement in neointimal hyperplasia. Circulation. 2000; 102: 1697–1702.
Jovinge S, Hultgardh-Nilsson A, Regnstrom J, Nilsson J. Tumor necrosis factor-α activates smooth muscle cell migration in culture and is expressed in the balloon-injured rat aorta. Arterioscler Thromb Vasc Biol. 1997; 17: 490–497.
Sheng M, Thompson MA, Greenberg ME. CREB: a Ca(2+)-regulated transcription factor phosphorylated by calmodulin-dependent kinases. Science. 1991; 252: 1427–1430.
Xing J, Ginty DD, Greenberg ME. Coupling of the RAS-MAPK pathway to gene activation by RSK2, a growth factor-regulated CREB kinase. Science. 1996; 273: 959–963.
Du K, Montminy M. CREB is a regulatory target for the protein kinase Akt/PKB. J Biol Chem. 1998; 273: 32377–32379.
Tokunou T, Ichiki T, Takeda K, Funakoshi Y, Iino N, Shimokawa H, Egashira K, Takeshita A. Thrombin induces interleukin-6 expression through the cAMP response element in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 2001; 21: 1759–1763.
Funakoshi Y, Ichiki T, Ito K, Takeshita A. Induction of interleukin-6 expression by angiotensin II in rat vascular smooth muscle cells. Hypertension. 1999; 34: 118–125.
Goetze S, Xi XP, Kawano Y, Kawano H, Fleck E, Hsueh WA, Law RE. TNF-α-induced migration of vascular smooth muscle cells is MAPK dependent. Hypertension. 1999; 33: 183–189.
Tokunou T, Shibata R, Kai H, Ichiki T, Morisaki T, Fukuyama K, Ono H, Iino N, Masuda S, Shimokawa H, Egashira K, Imaizumi T, Takeshita A. Apoptosis induced by inhibition of cyclic AMP response element-binding protein in vascular smooth muscle cells. Circulation. 2003; 108: 1246–1252.
Horwitz AR, Parsons JT. Cell migration—movin’ on. Science. 1999; 286: 1102–1103.
Hall A. Rho GTPases and the actin cytoskeleton. Science. 1998; 279: 509–514.
Nobes CD, Hall A. Rho GTPases control polarity, protrusion, and adhesion during cell movement. J Cell Biol. 1999; 144: 1235–1244.
Ryu Y, Takuwa N, Sugimoto N, Sakurada S, Usui S, Okamoto H, Matsui O, Takuwa Y. Sphingosine-1-phosphate, a platelet-derived lysophospholipid mediator, negatively regulates cellular Rac activity and cell migration in vascular smooth muscle cells. Circ Res. 2002; 90: 325–332.
Murphy AM, Montell DJ. Cell type-specific roles for Cdc42, Rac, and RhoL in Drosophila oogenesis. J Cell Biol. 1996; 133: 617–630.
Hagiwara M, Brindle P, Harootunian A, Armstrong R, Rivier J, Vale W, Tsien R, Montminy MR. Coupling of hormonal stimulation and transcription via the cyclic AMP-responsive factor CREB is rate limited by nuclear entry of protein kinase A. Mol Cell Biol. 1993; 13: 4852–4859.
Anderson MG, Dynan WS. Quantitative studies of the effect of HTLV-I Tax protein on CREB protein–DNA binding. Nucleic Acids Res. 1994; 22: 3194–3201.
Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science. 1995; 270: 1326–1331.
Huot J, Houle F, Marceau F, Landry J. Oxidative stress-induced actin reorganization mediated by the p38 mitogen-activated protein kinase/heat shock protein 27 pathway in vascular endothelial cells. Circ Res. 1997; 80: 383–392.
Matsumoto T, Yokote K, Tamura K, Takemoto M, Ueno H, Saito Y, Mori S. Platelet-derived growth factor activates p38 mitogen-activated protein kinase through a Ras-dependent pathway that is important for actin reorganization and cell migration. J Biol Chem. 1999; 274: 13954–13960.
Hedges JC, Dechert MA, Yamboliev IA, Martin JL, Hickey E, Weber LA, Gerthoffer WT. A role for p38(MAPK)/HSP27 pathway in smooth muscle cell migration. J Biol Chem. 1999; 274: 24211–24219.
Deak M, Clifton AD, Lucocq LM, Alessi DR. Mitogen- and stress-activated protein kinase-1 (MSK1) is directly activated by MAPK and SAPK2/p38, and may mediate activation of CREB. EMBO J. 1998; 17: 4426–4441.
Miki H, Suetsugu S, Takenawa T. WAVE, a novel WASP-family protein involved in actin reorganization induced by Rac. EMBO J. 1998; 17: 6932–6941.
Takenawa T, Miki H. WASP and WAVE family proteins: key molecules for rapid rearrangement of cortical actin filaments and cell movement. J Cell Sci. 2001; 114: 1801–1809.
Weih F, Stewart AF, Boshart M, Nitsch D, Schutz G. In vivo monitoring of a cAMP-stimulated DNA-binding activity. Genes Dev. 1990; 4: 1437–1449.
Klemm DJ, Watson PA, Frid MG, Dempsey EC, Schaack J, Colton LA, Nesterova A, Stenmark KR, Reusch JE. cAMP response element-binding protein content is a molecular determinant of smooth muscle cell proliferation and migration. J Biol Chem. 2001; 276: 46132–46141.
Tabernero A, Stewart HJ, Jessen KR, Mirsky R. The neuron-glia signal beta neuregulin induces sustained CREB phosphorylation on Ser-133 in cultured rat Schwann cells. Mol Cell Neurosci. 1998; 10: 309–322.
Pugazhenthi S, Boras T, O’Connor D, Meintzer MK, Heidenreich KA, Reusch JE. Insulin-like growth factor I-mediated activation of the transcription factor cAMP response element-binding protein in PC12 cells. Involvement of p38 mitogen-activated protein kinase-mediated pathway. J Biol Chem. 1999; 274: 2829–2837.
Funakoshi Y, Ichiki T, Takeda K, Tokuno T, Iino N, Takeshita A. Critical role of cAMP-response element-binding protein for angiotensin II-induced hypertrophy of vascular smooth muscle cells. J Biol Chem. 2002; 277: 18710–18717.