CREB cAMP Response Element-binding Protein Research Articles

BDNF- and VEGF-Responsive Stimulus to an NGF Mimic Cyclic Peptide with Copper Ionophore Capability and Ctr1/CCS-Driven Signaling.

Neurotrophins are a family of growth factors that play a key role in the development and regulation of the functioning of the central nervous system. Their use as drugs is made difficult by their poor stability, cellular permeability, and side effects. Continuing our effort to use peptides that mimic the neurotrophic growth factor (NGF), the family model protein, and specifically the N-terminus of the protein, here we report on the spectroscopic characterization and resistance to hydrolysis of the 14-membered cyclic peptide reproducing the N-terminus sequence (SSSHPIFHRGEFSV (c-NGF(1-14)). Far-UV CD spectra and a computational study show that this peptide has a rigid conformation and left-handed chirality typical of polyproline II that favors its interaction with the D5 domain of the NGF receptor TrkA. c-NGF(1-14) is able to bind Cu2+ with good affinity; the resulting complexes have been characterized by potentiometric and spectroscopic measurements. Experiments on PC12 cells show that c-NGF(1-14) acts as an ionophore, influencing the degree and the localization of both the membrane transporter (Ctr1) and the copper intracellular transporter (CCS). c-NGF(1-14) induces PC12 differentiation, mimics the protein in TrkA phosphorylation, and activates the kinase cascade, inducing Erk1/2 phosphorylation. c-NGF(1-14) biological activities are enhanced when the peptide interacts with Cu2+ even with the submicromolar quantities present in the culture media as demonstrated by ICP-OES measurements. Finally, c-NGF(1-14) and Cu2+ concur to activate the cAMP response element-binding protein CREB that, in turn, induces the brain-derived neurotrophic factor (BDNF) and the vascular endothelial growth factor (VEGF) release.

Exploring the Role of Asp1116 in Selective Drug Targeting of CREBcAMP- Responsive Element-binding Protein Implicated in Prostate Cancer.

The selective targeting of CREB-cAMP-responsive element-binding protein (CBP) has recently evolved as a vital therapeutic approach for curtailing its aberrant upregulation associated with the development of prostate cancer. Inhibition of CBP has been discovered to be an important therapeutic option in androgen receptor signalling pathway mediated prostate cancer. Y08197, a novel selective inhibitor of CBP, has shown promising therapeutic outcome in prostate carcinogenesis over non-selective analogues such as CPI-637. Herein, we used molecular dynamics simulation to gain insights into the mechanistic and selective targeting of Y08197 at the bromodomain active site. Molecular Mechanics/ Poisson-Boltzmann Surface Area (MM/PBSA) analysis revealed a similar inhibitory effect between Y08197 and CPI-637. Furthermore, in exploring the selective affinity of Y08197 towards CBP in combination with Bromodomain and PHD finger-containing protein 1(BRPF1), our findings highlighted Asp1116 as the 'culprit' residue responsible for this selective targeting. Upon binding, Asp1116 assumed a conformation that altered the architecture of the bromodomain active site, thereby orienting the helices around the active site in a more compacted position. In addition to some specific structural perturbations mediated by Asp1116 on the dynamics of CBP, our study revealed that the strong hydrogen bond interaction (N-H...O) elicited in CBP-Y08197 sequestered Y08197 tightly into the CBP bromodomain active site. Conclusively, the inhibition and selective pattern of Y08197 can be replicated in future structure-based CBP inhibitors and other bromodomain implicated in carcinogenesis.

Linking CREB function with altered metabolism in murine fibroblast-based model cell lines.

The cAMP-responsive element binding protein CREB is frequently overexpressed and activated in tumors of distinct histology, leading to enhanced proliferation, migration, invasion and angiogenesis as well as reduced apoptosis. The de-regulated expression of CREB might be linked with transcriptional as well as post-transcriptional regulation mechanisms. We show here that altered CREB expression levels and function are associated with changes in the cellular metabolism. Using comparative proteome-based analysis an altered expression pattern of proteins involved in the cellular metabolism in particular in glycolysis was found upon CREB down-regulation in HER-2/neu-transfected cell lines. This was associated with diminished expression levels of the glucose transporter 1, reduced glucose uptake and reduced glycolytic activity in HER-2/neu-transfected cells with down-regulated CREB when compared to HER-2/neu+ cells. Furthermore, hypoxia-induced CREB activity resulted in changes of the metabolism in HER-2/neu transfected cells. Low pH values in the supernatant of HER-2/neu transformants were restored by CREB down-regulation, but further decreased by hypoxia. The altered intracellular pH values were associated with a distinct expression of lactate dehydrogenase, and its substrate lactate. Moreover, enhanced phosphorylation of CREB on residue Ser133 was accompanied by a down-regulation of pERK and an up-regulation of pAKT. CREB promotes the detoxification of ROS by catalase, therefore protecting the mitochondrial activity under oxidative stress. These data suggest that there might exists a link between CREB function and the altered metabolism in HER-2/neu-transformed cells. Thus, targeting these altered metabolic pathways might represent an attractive therapeutic approach at least for the treatment of patients with HER-2/neu overexpressing tumors.

Silencing of ICERs (Inducible cAMP Early Repressors) results in partial protection of neurons from programmed cell death

ICERs proteins (Inducible cAMP Early Repressors) are the most effective endogenous repressors of CREB/CREM/ATF transcription factors family (CREB-cAMP Responsive Element Binding protein, CREM-cAMP Responsive Element Modulator, ATF-Activating Transcription Factor) that have repeatedly been shown to have a prosurvival function. It has been reported previously that neuronal death is accompanied by increased expression of ICERs and, furthermore, their overexpression provokes neuronal cell death in culture. However, it was not explained whether endogenously activated by proapoptotic stimuli ICERs contribute to the neuronal cell death. Herein, we have examined the involvement of endogenous ICERs in the apoptosis by checking whether it is possible to protect neurons from cell death by blocking the ICER gene. We applied two different in vitro models of neuronal death of primary neuronal cultures: excitotoxic death of neurons derived from dentate gyrus, and cortical cell loss provoked by trophic deprivation. Using the lentiviral vector (LV) to deliver shRNA, specifically silencing ICERs, but not other CREM proteins, we have found that silencing of ICERs enhances the CRE-driven transcription and exerts a mild, although significant, neuroprotective effect in both models. Since we demonstrated that silencing of endogenous ICERs have protective effect on neurons exposed to apoptosis-provoking conditions, targeting ICERs might be a novel strategy to prevent neuronal loss during degenerative processes.

A-kinase-interacting Protein 1 (AKIP1) Acts as a Molecular Determinant of PKA in NF-κB Signaling

The cAMP-dependent protein kinase (PKA) signaling pathway plays a crucial role in the pathogenesis of many NF-κB-related diseases. However, there have been controversial reports with regard to the PKA actions in the regulation of NF-κB activity. In this study, we have demonstrated the effect of PKA on NF-κB activity in view of AKIP1 action; and in 293 and HeLa cells, where the endogenous AKIP1 expression is minimal, PKA-activating agents inhibited the NF-κB-dependent reporter gene expression, blocked the interaction of PKAc and p65 subunit of NF-κB, and attenuated PKA-dependent phosphorylation of p65 on Ser-276. This inhibitory function of PKAc in NF-κB signaling was reversed by overexpression of AKIP1 in 293 cells. In the breast cancer cell line, MDA-MB231 cells and MCF7 cells, where the endogenous AKIP1 is abundant, the PKA signal was found to be synergized with NF-κB activation; PKA-activating agents enhanced NF-κB-dependent transcriptional activity and the interaction between p65 and PKAc and augmented the phosphorylation of p65 on Ser-276. After RNAi knockdown of AKIP1 in these breast cancer cells, we observed that PKA-activating agents antagonized NF-κB-dependent activation. Meanwhile, PKA inhibitor suppressed NF-κB-induced breast cancer cell proliferation and multiple NF-κB-dependent anti-apoptotic gene expression. It is likely that expression of AKIP1 determines the relationship between these two signal transduction pathways. These findings explained controversial results from various independent groups regarding the action of PKA signaling on the NF-κB activation cascade and suggested a possible therapeutic potential of PKA inhibitor in developing anti-cancer strategies. The cAMP-dependent protein kinase (PKA) signaling pathway plays a crucial role in the pathogenesis of many NF-κB-related diseases. However, there have been controversial reports with regard to the PKA actions in the regulation of NF-κB activity. In this study, we have demonstrated the effect of PKA on NF-κB activity in view of AKIP1 action; and in 293 and HeLa cells, where the endogenous AKIP1 expression is minimal, PKA-activating agents inhibited the NF-κB-dependent reporter gene expression, blocked the interaction of PKAc and p65 subunit of NF-κB, and attenuated PKA-dependent phosphorylation of p65 on Ser-276. This inhibitory function of PKAc in NF-κB signaling was reversed by overexpression of AKIP1 in 293 cells. In the breast cancer cell line, MDA-MB231 cells and MCF7 cells, where the endogenous AKIP1 is abundant, the PKA signal was found to be synergized with NF-κB activation; PKA-activating agents enhanced NF-κB-dependent transcriptional activity and the interaction between p65 and PKAc and augmented the phosphorylation of p65 on Ser-276. After RNAi knockdown of AKIP1 in these breast cancer cells, we observed that PKA-activating agents antagonized NF-κB-dependent activation. Meanwhile, PKA inhibitor suppressed NF-κB-induced breast cancer cell proliferation and multiple NF-κB-dependent anti-apoptotic gene expression. It is likely that expression of AKIP1 determines the relationship between these two signal transduction pathways. These findings explained controversial results from various independent groups regarding the action of PKA signaling on the NF-κB activation cascade and suggested a possible therapeutic potential of PKA inhibitor in developing anti-cancer strategies. NF-κB is an inducible transcriptional factor for the expression of multiple genes involved in immunoinflammatory responses, cell proliferation, and survival, thus playing crucial roles in the pathogenesis of many diseases, including cancer, leukemia, and autoimmune diseases (1Baeuerle P.A. Baichwal V.R. Adv. Immunol. 1997; 65: 111-137Crossref PubMed Google Scholar, 2Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5578) Google Scholar, 3Okamoto T. Sakurada S. Yang J.P. Merin J.P. Curr. Top. Cell. Regul. 1997; 35: 149-161Crossref PubMed Scopus (75) Google Scholar, 4Moynagh P.N. J. Cell Sci. 2005; 118: 4589-4592Crossref PubMed Scopus (335) Google Scholar). NF-κB primarily exists as the heterodimer consisting of p65 and p50 in the cytoplasm and is sequestered by binding to its inhibitory protein IκB (5Sun S.C. Ganchi P.A. Béraud C. Ballard D.W. Greene W.C. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 1346-1350Crossref PubMed Scopus (162) Google Scholar). Upon signaling, such as by tumor necrosis factor α (TNFα), IκB is phosphorylated and is followed by proteasome-mediated degradation, which liberates NF-κB to the nucleus thereby activating the target genes. PKA exists in the cytoplasm as an inactivated tetramer holoenzyme composed of dimer catalytic subunits and dimer regulatory subunits, which dissociate upon elevation of cAMP (6Carnegie G.K. Scott J.D. Genes Dev. 2003; 17: 1557-1568Crossref PubMed Scopus (56) Google Scholar, 7Taylor S.S. Buechler J.A. Yonemoto W. Annu. Rev. Biochem. 1990; 59: 971-1005Crossref PubMed Scopus (956) Google Scholar, 8Scott J.D. Pharmacol. Ther. 1991; 50: 123-145Crossref PubMed Scopus (314) Google Scholar, 9Francis S.H. Corbin J.D. Annu. Rev. Physiol. 1994; 56: 237-272Crossref PubMed Scopus (408) Google Scholar, 10Walsh D.A. Van Patten S.M. FASEB J. 1994; 8: 1227-1236Crossref PubMed Scopus (214) Google Scholar). PKAc is also predominantly involved in the IκB-NF-κB complex in the cytoplasm, and IκB sequesters PKAc by masking its catalytic domain (11Thompson J.E. Phillips R.J. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1995; 80: 573-582Abstract Full Text PDF PubMed Scopus (693) Google Scholar). Following a variety of extracellular stimuli such as TNFα, IκB is phosphorylated by IKK 2The abbreviations used are: IKKIκB kinaseBt2cAMPas dibutyryl cyclic AMPIP-WBimmunoprecipitation-Western blottingCREBcAMP-response element-binding proteinCBPCREB binding protein. and degraded by the 26 S proteasome (12Lallena M.J. Diaz-Meco M.T. Bren G. Payá C.V. Moscat J. Mol. Cell. Biol. 1999; 19: 2180-2188Crossref PubMed Google Scholar, 13DiDonato J. Mercurio F. Rosette C. Wu-Li J. Suyang H. Ghosh S. Karin M. Mol. Cell. Biol. 1996; 16: 1295-1304Crossref PubMed Google Scholar). As a consequence, the p65/p50 heterodimer complex is liberated, and the catalytic center of PKAc is exposed, enabling activated PKAc to phosphorylate p65 at Ser-276 (14Zhong H. SuYang H. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1997; 89: 413-424Abstract Full Text Full Text PDF PubMed Scopus (727) Google Scholar, 15Zhong H. Voll R.E. Ghosh S. Mol. Cell. 1998; 1: 661-671Abstract Full Text Full Text PDF PubMed Scopus (1023) Google Scholar). This PKA-dependent phosphorylation of p65 facilitates the recruitment of transcription coactivator CBP and DNA binding activity of p65; therefore, activation of PKA augments NF-κB-dependent gene expression, and PKAc signaling is considered to up-regulate NF-κB activation (14Zhong H. SuYang H. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1997; 89: 413-424Abstract Full Text Full Text PDF PubMed Scopus (727) Google Scholar, 15Zhong H. Voll R.E. Ghosh S. Mol. Cell. 1998; 1: 661-671Abstract Full Text Full Text PDF PubMed Scopus (1023) Google Scholar, 16Hayashi T. Sekine T. Okamoto T. J. Biol. Chem. 1993; 268: 26790-26795Abstract Full Text PDF PubMed Google Scholar, 17Chen Lf. Fischle W. Verdin E. Greene W.C. Science. 2001; 293: 1653-1657Crossref PubMed Scopus (1049) Google Scholar). With regard to the role of PKAc in NF-κB signaling, some claimed that cAMP-dependent PKA activation down-regulated NF-κB-dependent transcription by changing its DNA binding ability (18Kim Y.S. Cho K.O. Lee H.J. Kim S.Y. Sato Y. Cho Y.J. J. Biol. Chem. 2006; 281: 39051-39061Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 19Neumann M. Grieshammer T. Chuvpilo S. Kneitz B. Lohoff M. Schimpl A. Franza Jr., B.R. Serfling E. EMBO J. 1995; 14: 1991-2004Crossref PubMed Scopus (181) Google Scholar), modifying the transactivation domain of p65 (20Takahashi N. Tetsuka T. Uranishi H. Okamoto T. Eur. J. Biochem. 2002; 269: 4559-4565Crossref PubMed Scopus (131) Google Scholar), or blocking the degradation of IκB proteins (21Majumdar S. Aggarwal B.B. Oncogene. 2003; 22: 1206-1218Crossref PubMed Scopus (90) Google Scholar, 22Minguet S. Huber M. Rosenkranz L. Schamel W.W. Reth M. Brummer T. Eur. J. Immunol. 2005; 35: 31-41Crossref PubMed Scopus (165) Google Scholar). IκB kinase as dibutyryl cyclic AMP immunoprecipitation-Western blotting cAMP-response element-binding protein CREB binding protein. In our previous report, we identified AKIP1 as a novel p65-interacting protein (23Gao N. Asamitsu K. Hibi Y. Ueno T. Okamoto T. J. Biol. Chem. 2008; 283: 7834-7843Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). AKIP1 was initially found in breast cancer cells (24Kitching R. Li H. Wong M.J. Kanaganayakam S. Kahn H. Seth A. Biochim. Biophys. Acta. 2003; 1625: 116-121Crossref PubMed Scopus (41) Google Scholar) in which it facilitated the nuclear translocation of PKAc (25Sastri M. Barraclough D.M. Carmichael P.T. Taylor S.S. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 349-354Crossref PubMed Scopus (108) Google Scholar). We found that AKIP1 appears to serve as a molecular bridge between p65 and PKAc, promoting their interaction and subsequent p65 phosphorylation at Ser-276, thus enhancing NF-κB signaling (23Gao N. Asamitsu K. Hibi Y. Ueno T. Okamoto T. J. Biol. Chem. 2008; 283: 7834-7843Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Because NF-κB is constitutively activated in some breast cancer cells, endowing them with resistance to apoptosis (26Kajino S. Suganuma M. Teranishi F. Takahashi N. Tetsuka T. Ohara H. Itoh M. Okamoto T. Oncogene. 2000; 19: 2233-2239Crossref PubMed Scopus (34) Google Scholar, 27Sanda T. Iida S. Ogura H. Asamitsu K. Murata T. Bacon K.B. Ueda R. Okamoto T. Clin. Cancer Res. 2005; 11: 1974-1982Crossref PubMed Scopus (66) Google Scholar, 28Specht K. Haralambieva E. Bink K. Kremer M. Mandl-Weber S. Koch I. Tomer R. Hofler H. Schuuring E. Kluin P.M. Fend F. Quintanilla-Martinez L. Blood. 2004; 104: 1120-1126Crossref PubMed Scopus (98) Google Scholar), we hypothesized that the effect of PKA in NF-κB cascade is associated with AKIP1 and could be modulated by AKIP1 in a cell type-dependent fashion. In this study, we further investigate the role of PKA in regulating NF-κB-dependent transcription in various cell lines with different expression levels of endogenous AKIP1. We provide evidence that in minimal AKIP1-expressing cell lines, elevation of cAMP decreased p65-PKA binding and p65 phosphorylation at Ser-276, which eventually leads to down-regulation of the NF-κB-dependent transcription. In contrast, in breast cancer cell lines MDA-MB231 and MCF7 with high AKIP1 expression, the PKA-activating agents increased p65-PKA binding and its phosphorylation and up-regulated the NF-κB-dependent transcription, particularly in anti-apoptotic genes. 293, HeLa, MCF7, and MDA-MB231 cells (ATCC) were cultured at 37 °C with 5% CO2 in Dulbecco's modified Eagle's medium with 10% heat-inactivated fetal bovine serum, 100 units of penicillin, and 100 μg/ml streptomycin. Cells were transfected using FuGENE 6 transfection reagent (Roche Applied Science) according to the manufacturer's recommendations. Cell lysates from 293, HeLa, MCF7, and MDA-MB231 cells were collected, and Western blotting assay was performed by using polyclonal antibody raised against AKIP1 (kindly provided by Professor S. Taylor) (25Sastri M. Barraclough D.M. Carmichael P.T. Taylor S.S. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 349-354Crossref PubMed Scopus (108) Google Scholar). The RNAi sequence of AKIP1 (UCC UCU UGG CCC UCU CCA GCA CUUC) was designed from exon 2 of AKIP1 (amino acids 155–179) to degrade all endogenous AKIP1, including its splicing variants, including AKIP1b and AKIP1c (23Gao N. Asamitsu K. Hibi Y. Ueno T. Okamoto T. J. Biol. Chem. 2008; 283: 7834-7843Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). These RNAi reagents for AKIP1 were purchased from Invitrogen. Transfection of AKIP1 RNAi was performed using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's recommendations. After transfecting with or without FLAG-AKIP1, 293 cells were cultured with the cAMP analogue Bt2cAMP (3 mm) for 12 h and TNFα (1 ng/ml) for an additional 6 h. Immunoprecipitation was performed as described previously (23Gao N. Asamitsu K. Hibi Y. Ueno T. Okamoto T. J. Biol. Chem. 2008; 283: 7834-7843Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) by using either anti-p65 antibody or anti-CREB antibody (Santa Cruz Biotechnology), and incubated with protein A-Sepharose beads (Sigma). Bound proteins were eluted with an equal volume of SDS loading buffer at 100 °C for 3 min and fractionated on 10% SDS-PAGE. Western blotting was conducted using either anti-PKAc (Santa Cruz Biotechnology), anti-p65-phospho-Ser-276 (Cell Signaling), anti-CBP (Cell Signaling), or anti-CREB-phospho-Ser-133 (Cell Signaling) antibodies. MDA-MB231 cells with or without AKIP1 knockdown by RNAi (Invitrogen) were also used for the same IP-WB assays. HeLa, 293, MCF7, and MDA-MB231 cells were cultured in 24-well plates, and FLAG-AKIP1 expression plasmid together with pGL-3κB-luc and pRL-TK plasmids were transfected with FuGENE 6 transfection reagent (Roche Applied Science). 24 h after transfection, cells were treated with Bt2cAMP or Br-cAMP (1 mm, 3 mm) for 12 h and subsequently stimulated with TNFα (1 ng/ml) for an additional 6 h. These cells were harvested for luciferase assay. Luciferase activity was normalized by the Renilla luciferase activity of each transfection to normalize the transfection efficiency. ELISAs were performed using commercial IL-8 ELISA kits (R & D Systems). Culture supernatants from 293 cells and MDA-MB231 cells with or without Bt2cAMP/TNFα treatment were collected for the IL-8 concentration determination. Assays were carried out in triplicate, and readings were compared with a standard curve obtained with human recombinant IL-8 provided in the kit. MDA-MB231 and 293 cells were seeded in 96-well plates, incubated for 24 h, treated with the PKA inhibitor H89 (1 and 3 μm) for 4 h, and further incubated with the IKK inhibitor BAY11-7082 (5, 10, and 20 μm) for an additional 20 h. WST-1 assay was performed by adding 10 μl of WST-1 reagent (Roche Applied Science) to each sample and incubated for 1 h at 37 °C. The absorbance of each sample was then measured at 450 nm. 293 cells were cultured in 6-well plates with or without AKIP1 transduction and were treated with or without Bt2cAMP (3 mm) and/or TNFα (1 ng/ml). Total RNA was isolated using TRIzol reagent (Invitrogen), and 0.3 μg total RNA of each sample was reverse-transcribed into cDNA. Real time PCR was carried out with quantitative PCR MasterMix Plus for SYBR Green using the instruction manual: incubation at 95 °C for 10 min followed by a 40-cycle amplification (15 s at 95 °C, 1 min at 60 °C, 45 s at 72 °C, and 15 s at 80 °C) for SYBR Green detection. Amplification was performed with the following primers: cyclin D1, sense 5′-CCC TCG GTG TCC TAC TTC AAA-3′ and antisense 5′-CAC CTC CTC CTC CTC CTC TTC-3′; bcl-2, sense 5′-AGA TGT CCA GGC AGC TGC AC-3′ and antisense 5′-CAC AGG GCG ATG TTG TCC AC-3′; bcl-xL, sense 5′- CAG AGC AAC CGG GAG CTG GT-3′ and antisense 5′-GAT CCA AGG CTC TAG GTG GTC-3′; survivin, sense 5′- GGC ATG GGT GCC CCG ACG TTG-3′ and antisense 5′-CAG AGG CCT CAA TCC ATG GCA-3′; xiap, sense 5′-TGT CCG ATG TGC AAC ACA GT-3′ and antisense 5′- CCA TGT CAG TAC ATG TAG GC-3′; and β-actin, sense CCA GGC ACC AGG GCG TGA TG-3′ and antisense 5′-CGG CCA GCC AGG TCC AGA CG-3′ (27Sanda T. Iida S. Ogura H. Asamitsu K. Murata T. Bacon K.B. Ueda R. Okamoto T. Clin. Cancer Res. 2005; 11: 1974-1982Crossref PubMed Scopus (66) Google Scholar). The expression of each gene was normalized by the corresponding amount of β-actin mRNA. The relative amounts of each product were calculated by the comparative CT (2−ΔΔCT) method described in User Bulletin 2 of the ABI Prism 7500 Fast Sequence Detection System (Applied Biosystems). To elucidate the role of AKIP1, a newly identified binding partner of the p65 subunit of NF-κB, we first examined its expression in various cell lines. Endogenous AKIP1 levels were detected by Western blot assay using a specific anti-AKIP1 antibody (25Sastri M. Barraclough D.M. Carmichael P.T. Taylor S.S. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 349-354Crossref PubMed Scopus (108) Google Scholar). As shown in Fig. 1, expression of endogenous AKIP1 was very low in 293 and HeLa cells, whereas in breast cancer cells MDA-MB231 and MCF7, AKIP1 was abundantly expressed. This result was consistent with the previous report indicating AKIP1 is highly expressed in breast cancer cells (24Kitching R. Li H. Wong M.J. Kanaganayakam S. Kahn H. Seth A. Biochim. Biophys. Acta. 2003; 1625: 116-121Crossref PubMed Scopus (41) Google Scholar). It is also important to note that the highest AKIP1 expression was observed in MDA-MB231, which was derived from metastasized breast cancer tissue and was considered highly malignant (29Bendre M.S. Gaddy-Kurten D. Mon-Foote T. Akel N.S. Skinner R.A. Nicholas R.W. Suva L.J. Cancer Res. 2002; 62: 5571-5579PubMed Google Scholar). Considering biological effects and regulation are mediated through protein-protein interaction, and it was established that PKAc is involved in regulating p65 phosphorylation at Ser-276, we attempted to explore the p65-PKAc interaction in various cell lines with different levels of AKIP1 expression. In Fig. 2A, 293 cells exhibiting low expression levels of endogenous AKIP1 were treated with the PKAc activator Bt2cAMP and NF-κB inducer TNFα. These cells were then subjected to immunoprecipitation with anti-p65 antibody to detect p65-PKAc binding through Western blotting using anti-PKAc antibody. As demonstrated here, treatment with Bt2cAMP enhanced the p65-PKAc interaction (Fig. 2A, upper panel, lane 2), suggesting cAMP could activate the PKAc-p65 interaction even without TNFα. Upon stimulation with TNFα, the interaction between p65 and PKAc was similarly enhanced (Fig. 2A, upper panel, lane 3). Intriguingly, treatment with both Bt2cAMP and TNFα at the same time led to a relatively weaker binding between p65 and PKA as compared with the treatment with TNFα alone (Fig. 2A, upper panel, compare lane 3 with 4), suggesting that the combination of PKA and NF-κB signaling could rather inhibit the p65-PKA interaction. According to our previous study, AKIP1 could enhance NF-κB activation by promoting PKAc-dependent p65 phosphorylation at Ser-276 in most cell lines (23Gao N. Asamitsu K. Hibi Y. Ueno T. Okamoto T. J. Biol. Chem. 2008; 283: 7834-7843Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Here, we examined whether there is a correlation between the extent of PKA-mediated p65 phosphorylation and the amount of AKIP1 expression. As shown in Fig. 2A (middle panel), when 293 cells were precipitated by anti-p65 antibody, and immunoblotted with anti-phospho-p65 (Ser-276) antibody, p65 phosphorylation was induced either by Bt2cAMP (lane 2) or TNFα (lane 3). However, when these cells were treated with both TNFα and Bt2cAMP, the extent of p65 phosphorylation and of p65-PKAc interaction was diminished (Fig. 2A, lane 4). To examine the action of AKIP1 in regulating PKA signaling in the NF-κB activation pathway, we transfected the AKIP1-expressing plasmid in 293 cells, and the IP-WB assay was performed with p65 antibody followed by PKAc antibody. As shown in Fig. 2B (upper panel), p65-PKAc binding was moderately enhanced by Bt2cAMP (lane 2) and by TNFα (lane 3). Surprisingly, when AKIP1 was overexpressed, treatment of both Bt2cAMP and TNFα significantly increased p65-PKAc interaction (compare lane 4 of Fig. 2A with that of B, upper panels). Similarly, the extent of p65 (Ser-276) phosphorylation was further enhanced by both Bt2cAMP and TNFα. These results suggested that AKIP1 could reverse the inhibitory function of PKAc in antagonizing NF-κB cascade. We investigated the effect of endogenous AKIP1 in regulating the enzymatic action of PKAc (Fig. 2, C and D). Either AKIP1-expressing breast cancer cell MDA-MB231 or AKIP1-knockdown MDA-MB231 cells were subjected to IP-WB assay with anti-p65 antibody followed by anti-PKAc antibody and phospho-p65 (Ser-276) antibody. Western blot assay revealed the significant degradation of endogenous AKIP1 protein after transfecting RNAi against AKIP1 in MDA-MB231 cells (data not shown). As shown in Fig. 2C (upper panel) with MDA-MB231 cells expressing endogenous AKIP1, either Bt2cAMP or TNFα could elevate the interaction between p65 and PKAc (lanes 2 and 3). Furthermore, upon the treatment with both Bt2cAMP and TNFα, p65-PKAc binding was up-regulated by PKAc activation (Fig. 2C, lane 4). Activation of PKAc induced an increased p65 phosphorylation at Ser-276 (Fig. 2C, middle panel, lane 2), and upon stimulation with TNFα, p65 phosphorylation was enhanced to the similar level (lane 3). When these cells were treated with both Bt2cAMP and TNFα, the p65 Ser-276 phosphorylation was further augmented (Fig. 2C, lane 4). These results mimic the observations with AKIP1-overexpressed 293 cells (Fig. 2B). Fig. 2D indicated that in AKIP1-knockdown MDA-MB231 cells, PKAc activator inhibited both the extent of p65-PKA interaction and PKA-mediated p65 phosphorylation upon TNFα treatment, thus mimicking the property of 293 cells (Fig. 2A). Overall, these observations in Fig. 2 clearly indicate that AKIP1 could alter the p65-PKAc interaction and subsequent p65 phosphorylation; in the presence of AKIP1, PKAc activation promoted p65-PKAc binding and PKA-mediated p65 phosphorylation, whereas in the absence of AKIP1, PKAc activator blocked p65-PKAc interaction and p65 phosphorylation. It is well established that PKA-mediated phosphorylation facilitates p65 to recruit its transcription coactivator, CBP (CREB-binding protein) (14Zhong H. SuYang H. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1997; 89: 413-424Abstract Full Text Full Text PDF PubMed Scopus (727) Google Scholar, 17Chen Lf. Fischle W. Verdin E. Greene W.C. Science. 2001; 293: 1653-1657Crossref PubMed Scopus (1049) Google Scholar). Here, we examined whether CBP recruitment could be affected by AKIP1 expression. In Fig. 3A, after 293 cells were treated with Bt2cAMP and/or TNFα, cell lysates were prepared, and an equal amount of p65 was immunoprecipitated with anti-p65 and blotted with anti-CBP antibodies. The interaction between p65 and CBP was increased upon treatment of either PKA activator or TNFα, but it was not enhanced when cells were treated both with Bt2cAMP and TNFα at the same time (compare lane 4 with lanes 2 and 3), which is consistent with the results in Fig. 2. We then hypothesized that other PKA-mediated transcription factors might be involved and disrupt PKA-p65 interaction. CREB, which is another PKA-dependent transcriptional factor, was reported to compete with p65 in recruiting CBP upon PKA stimulation (15Zhong H. Voll R.E. Ghosh S. Mol. Cell. 1998; 1: 661-671Abstract Full Text Full Text PDF PubMed Scopus (1023) Google Scholar, 16Hayashi T. Sekine T. Okamoto T. J. Biol. Chem. 1993; 268: 26790-26795Abstract Full Text PDF PubMed Google Scholar, 30Perkins N.D. Felzien L.K. Betts J.C. Leung K. Beach D.H. Nabel G.J. Science. 1997; 275: 523-527Crossref PubMed Scopus (666) Google Scholar). As shown in Fig. 3B, the cell lysate was prepared from the same 293 cells and immunoprecipitated by anti-CREB antibody, and then an equal amount of immune complex was blotted with antibodies against CBP and the phosphorylated CREB at Ser-133. With treatment with either Bt2cAMP or TNFα, the CREB-CBP interaction was significantly augmented. However, in contrast to p65-CBP binding, the interaction between CREB and CBP was enhanced even by treatment with both Bt2cAMP and TNFα (compare Fig. 3, A and B, lanes 4, upper panel). Furthermore, CREB phosphorylation was also increased (Fig. 3B, lane 4), consistent with the previous work that CREB recruits CBP upon phosphorylation of CREB by PKA (31De Cesare D. Fimia G.M. Sassone-Corsi P. Trends Biochem. Sci. 1999; 24: 281-285Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). In contrast, in AKIP1-overexpressing 293 cells (Fig. 3C), the combination of Bt2cAMP and TNFα induced the extent of p65-CBP interaction (Fig. 3C, upper panel, lane 4); however, CREB-CBP binding and CREB phosphorylation were significantly decreased (Fig. 3D, lane 4). Taken together, these results demonstrated in FIGURE 2, FIGURE 3 suggested that AKIP1 could modulate the relative preference for PKA-mediated phosphorylation and coactivator recruitment between p65 and CREB. AKIP1 attenuates the CREB phosphorylation and the subsequent binding to CBP, whereas AKIP1 appears to potentiate the recruitment of CBP to p65. The experiments described above indicated that AKIP1 appears to facilitate the PKA-dependent p65 phosphorylation and CBP recruitment upon TNFα induction. Thus, we examined whether AKIP1 could also regulate the effect of PKA in NF-κB-dependent transcriptional activity. As shown in Fig. 4A (left panel), in 293 cells (closed columns) where the expression of AKIP1 is limited, NF-κB-dependent gene expression was down-regulated by PKA activator Bt2cAMP in a dose-dependent manner. However, in MDA-MB231 cells (open columns), where AKIP1 is abundant, the NF-κB activation was up-regulated upon treatment with Bt2cAMP. We also tested another PKA activator Br-cAMP, and similar results were observed (data not shown). These results have demonstrated that AKIP1 determines the transcriptional activity of NF-κB through regulating the PKA signaling. To further address the effect of AKIP1 in mediating PKA and NF-κB signaling, we performed luciferase assays in 293 cells with or without AKIP1 overexpression. As shown in Fig. 4A (right panel), in wild type 293 cells, Bt2cAMP-mediated PKAc activation significantly repressed NF-κB-dependent transcription (closed columns), as in Fig. 4A (left panel). In contrast, in the AKIP1-overexpressing 293 cells, PKA activation augmented the NF-κB-dependent transcription in a dose-dependent manner (Fig. 4A, open columns). These results together with those in FIGURE 2, FIGURE 3 indicate that AKIP1 regulates the direction of cross-talk between PKA and NF-κB signaling presumably through the PKAc-mediated p65 phosphorylation and recruitment of the transcriptional coactivator CBP. Considering IL-8 is one of the physiological target genes governed by NF-κB and is closely involved in cancer progression through promoting cancer cell proliferation (32Xie K. Cytokine Growth Factor Rev. 2001; 12: 375-391Crossref PubMed Scopus (605) Google Scholar) and angiogenesis (33Shono T. Ono M. Izumi H. Jimi S.I. Matsushima K. Okamoto T. Kohno K. Kuwano M. Mol. Cell. Biol. 1996; 16: 4231-4239Crossref PubMed Google Scholar, 34Martin D. Galisteo R. Gutkind J.S. J. Biol. Chem. 2009; 284: 6038-6042Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar), we examined whether IL-8 expression is also regulated by AKIP1 through modifying the relationship between PKA and NF-κB signaling. As shown in Fig. 4B, 293 and MDA-MB231 cells were treated with Bt2cAMP and TNFα, and then the IL-8 concentration in cell culture supernatant was evaluated by sandwich-ELISA assay. In 293 cells, IL-8 production was triggered by TNFα stimulation, and in combination with Bt2cAMP, it was down-regulated in a dose-dependent manner (left panel). However, in MDA-MB231 cells, where the basic level of IL-8 is much higher than in 293 cells, the level of IL-8 production was further augmented by TNFα stimulation in concert with Bt2cAMP (Fig. 4B, right panel). Other PKA activators such as forskolin and Br-cAMP were also tested in these cell lines and MCF7 cells with similar results obtained (data not shown). These results indicated that NF-κB-dependent gene expression could be mediated by AKIP1 through PKA. This would all indicate that in the absence of AKIP1, PKA activation down-regulates NF-κB-dependent gene expression; however, in the presence of AKIP1, PKA activation up-regulates NF-κB-dependent gene expression. Aberrant activation of NF-κB signaling causes proliferation of cancer cells (2Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5578) Google Scholar, 3Okamoto T. Sakurada S. Yang J.P. Merin J.P. Curr. Top. Cell. Regul. 1997; 35: 149-161Crossref PubMed Scopus (75) Google Scholar). Because PKA activation synergizes with NF-κB cascade in breast cancer cells (Fig. 4), we hypothesized that PKA inhibitor would also down-regulate proliferation of breast cancer cells through the regulatory cascade of NF-κB activation. Treatment with H89, a PKA inhibitor, decreased the growth of MDA-MB231 cells in a dose-dependent manner (Fig. 5A). In addition, when BAY11-7082, a specific inhibitor of IκB kinase (35Mori N. Yamada Y. Ikeda S. Yamasaki Y. Tsukasaki K. Tanaka Y. Tomonaga M. Yamamoto N. Fujii M. Blood. 2002; 100: 1828-1834Crossref PubMed Scopus (255) Google Scholar, 36Dai Y. Pei X.Y. Rahmani M. Conrad D.H. Dent P. Grant S. Blood. 2004; 103: 2761-2770Crossref PubMed Scopus (93) Google Scholar), was added, the inhibitory effects of H89 were further potentiated (Fig. 5B). However, this synergism by IKK and PKA inhibitors was not observed in 293 cells (Fig. 5C), presumably because AKIP1 expression is minimum, and neither NF-κB nor PKA signaling is constitutively activated in 293 cells. IKK inhibitor BAY11-7082 could only repress the basal level of NF-κB activation, and PKA inhibitor H89 failed to further synergize with BAY11-7082. These observations suggest a possibility that H89 may require either IKK or PKA signaling that is associated with AKIP1 expression. Constitutive NF-κB activation induces expression of anti-apoptotic genes such as cyclin D1, bcl-2, bcl-xL, survivin, and xiap (26Kajino S. Suganuma M. Teranishi F. Takahashi N. Tetsuka T. Ohara H. Itoh M. Okamoto T. Oncogene. 2000; 19: 2233-2239Crossref PubMed Scopus (34) Google Scholar, 27Sanda T. Iida S. Ogura H. Asamitsu K. Murata T. Bacon K.B. Ueda R. Okamoto T. Clin. Cancer Res. 2005; 11: 1974-1982Crossref PubMed Scopus (66) Google Scholar, 28Specht K. Haralambieva E. Bink K. Kremer M. Mandl-Weber S. Koch I. Tomer R. Hofler H. Schuuring E. Kluin P.M. Fend F. Quintanilla-Martinez L. Blood. 2004; 104: 1120-1126Crossref PubMed Scopus (98) Google Scholar). We thus examined the effect of PKA activator Bt2cAMP on expression of these genes. Isolated total RNA from 293 cells with or without AKIP1 transfection and treatment of Bt2cAMP/TNFα were reverse-transcribed to cDNA. In wild type 293 cells (Fig. 6A), treatment of both TNFα and Bt2cAMP decreased the anti-apoptotic gene expression compared with TNFα-treated cells. However, among AKIP1-overexpressing 293 cells (Fig. 6B), double treatment of Bt2cAMP and TNFα enhanced the gene expression levels (lane 8). Consistent with our earlier results, AKIP1 appears to act as a major PKA determinant in the NF-κB activation cascade, and in cancer cells it promotes cell growth by up-regulating NF-κB and expression of its target genes. In this study, we investigated the cross-talk between two distinct pathways, namely NF-κB and PKA cascades with AKIP1 acting as a common interacting protein partner and co-factor for their actions. We found that AKIP1 could regulate the mode of PKA signaling on NF-κB cascade based on its expression level. In cell lines where AKIP1 expression was low, PKA activator inhibited p65-PKA binding and p65 phosphorylation (Fig. 2), accompanied with down-regulation of NF-κB-dependent transcription (Fig. 4). On the contrary, in cells like MDA-MB231 or AKIP1-overexpressing 293 cells, the p65-PKA binding and subsequent p65 phosphorylation were enhanced by PKA signaling (Fig. 2), thus up-regulating NF-κB-dependent transcription (Fig. 4). We also obtained similar results with nonmetastatic MCF7 breast cancer cells (data not shown). It is noticeable that when AKIP1 was transduced into 293 cells, PKA activator could augment NF-κB activation, which highlighted the significance of AKIP1 as a determinant of synergism between two cascades. In the presence of AKIP1 expression, PKA signaling augmented expression of anti-apoptotic genes thus contributing to the NF-κB-dependent cell survival (FIGURE 4, FIGURE 5, FIGURE 6). These findings give a clue to a long standing controversy regarding the action of PKA signaling in the NF-κB activation cascade (14Zhong H. SuYang H. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1997; 89: 413-424Abstract Full Text Full Text PDF PubMed Scopus (727) Google Scholar, 15Zhong H. Voll R.E. Ghosh S. Mol. Cell. 1998; 1: 661-671Abstract Full Text Full Text PDF PubMed Scopus (1023) Google Scholar, 16Hayashi T. Sekine T. Okamoto T. J. Biol. Chem. 1993; 268: 26790-26795Abstract Full Text PDF PubMed Google Scholar, 18Kim Y.S. Cho K.O. Lee H.J. Kim S.Y. Sato Y. Cho Y.J. J. Biol. Chem. 2006; 281: 39051-39061Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 19Neumann M. Grieshammer T. Chuvpilo S. Kneitz B. Lohoff M. Schimpl A. Franza Jr., B.R. Serfling E. EMBO J. 1995; 14: 1991-2004Crossref PubMed Scopus (181) Google Scholar, 20Takahashi N. Tetsuka T. Uranishi H. Okamoto T. Eur. J. Biochem. 2002; 269: 4559-4565Crossref PubMed Scopus (131) Google Scholar, 21Majumdar S. Aggarwal B.B. Oncogene. 2003; 22: 1206-1218Crossref PubMed Scopus (90) Google Scholar, 22Minguet S. Huber M. Rosenkranz L. Schamel W.W. Reth M. Brummer T. Eur. J. Immunol. 2005; 35: 31-41Crossref PubMed Scopus (165) Google Scholar);: the synergism or antagonism between PKA and NF-κB signals appear to depend on cell type and the level of AKIP1 expression in particular. It is well established that the recruitment of transcriptional coactivator CBP by p65 is an essential step for induction of its target genes by NF-κB (15Zhong H. Voll R.E. Ghosh S. Mol. Cell. 1998; 1: 661-671Abstract Full Text Full Text PDF PubMed Scopus (1023) Google Scholar, 30Perkins N.D. Felzien L.K. Betts J.C. Leung K. Beach D.H. Nabel G.J. Science. 1997; 275: 523-527Crossref PubMed Scopus (666) Google Scholar). Interestingly, PKA-mediated phosphorylation of p65 at Ser-276 promoted the interaction between p65 and CBP (Fig. 3). On the other hand, PKA also regulated the activity of CREB by phosphorylating on Ser-133 and promoting its binding to CBP (31De Cesare D. Fimia G.M. Sassone-Corsi P. Trends Biochem. Sci. 1999; 24: 281-285Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). Thus, two transcription factors appear to compete with each other in recruiting CBP, whose amount is known to be limited in cells (37Xu W. Chen H. Du K. Asahara H. Tini M. Emerson B.M. Montminy M. Evans R.M. Science. 2001; 294: 2507-2511Crossref PubMed Scopus (337) Google Scholar, 38Koh S.S. Li H. Lee Y.H. Widelitz R.B. Chuong C.M. Stallcup M.R. J. Biol. Chem. 2002; 277: 26031-26035Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). However, in the presence of AKIP1, such as in breast cancer cells, AKIP1 removes CBP from CREB and redirects it to p65, and AKIP1 seems to provide a molecular bridge for the interaction between PKAc and p65, inducing PKAc-mediated phosphorylation of p65 at Ser-276 thus endowing p65 with higher affinity to CBP (FIGURE 2, FIGURE 3). PKA activation is reported to positively regulate cancer progression through multiple cascades. For example, PKA activation induced osteolytic metastasis of MDA-MB231 breast cancer cells through ERK1/2-dependent pathway (39Shimo T. Kubota S. Yoshioka N. Ibaragi S. Isowa S. Eguchi T. Sasaki A. Takigawa M. J. Bone Miner. Res. 2006; 21: 1045-1059Crossref PubMed Scopus (135) Google Scholar). In addition, inhibition of PKA signaling induced apoptosis via down-regulation of Bcl2 expression (40Srivastava R.K. Srivastava A.R. Seth P. Agrawal S. Cho-Chung Y.S. Mol. Cell. Biochem. 1999; 195: 25-36Crossref PubMed Scopus (36) Google Scholar). Furthermore, the PKA inhibitor H89 could suppress breast cancer cell proliferation through blocking of the PKA-mediated STAT phosphorylation (41Xu H. Washington S. Verderame M.F. Manni A. Breast Cancer Res. Treat. 2008; 107: 63-70Crossref PubMed Scopus (6) Google Scholar). It was reported that PKA-mediated phosphorylation of either steroid receptor coactivator (Src1) or estrogen receptor itself endowed breast cancer cells with resistance to tamoxifen (42Michalides R. Griekspoor A. Balkenende A. Verwoerd D. Janssen L. Jalink K. Floore A. Velds A. van't Veer L. Neefjes J. Cancer Cell. 2004; 5: 597-605Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 43Zwart W. Griekspoor A. Berno V. Lakeman K. Jalink K. Mancini M. Neefjes J. Michalides R. EMBO J. 2007; 26: 3534-3544Crossref PubMed Scopus (96) Google Scholar). Moreover, inhibition of PKA pathway by H89 could abolish the cAMP-mediated aromatase induction through up-regulating brca1 expression (44Ghosh S. Lu Y. Hu Y. Int. J. Biomed. Sci. 2008; 4: 260-265PubMed Google Scholar). Similar to our study, a number of previous reports support the possibility that PKA signaling contributes to the constitutive NF-κB activation (45Arun P. Brown M.S. Ehsanian R. Chen Z. Van Waes C. Clin. Cancer Res. 2009; 15: 5974-5984Crossref PubMed Scopus (51) Google Scholar, 46Liu G.H. Qu J. Shen X. Biochim. Biophys. Acta. 2008; 1783: 713-727Crossref PubMed Scopus (485) Google Scholar, 47Ishinaga H. Jono H. Lim J.H. Kweon S.M. Xu H. Ha U.H. Xu H. Koga T. Yan C. Feng X.H. Chen L.F. Li J.D. EMBO J. 2007; 26: 1150-1162Crossref PubMed Scopus (80) Google Scholar). In this study, we demonstrated that NF-κB inhibitor BAY11-7082 repressed MDA-MB231 cell proliferation by blocking IKK, and PKA inhibitor H89 further augmented this inhibitory action (Fig. 5B). Thus, it is conceivable that PKA inhibitors would suppress the constitutive activation of NF-κB, potentiating the actions of NF-κB inhibitors. Potent inhibitors of PKA such as H89, PKI6-22 (23Gao N. Asamitsu K. Hibi Y. Ueno T. Okamoto T. J. Biol. Chem. 2008; 283: 7834-7843Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), and their derivatives could have potential therapeutic efficacy against cancer and leukemia. It is noticeable that PKA inhibitors could only block proliferation of breast cancer cells where AKIP1 is abundantly expressed but not in cells where it is absent (Fig. 5). Therefore, the expression level of AKIP1 may predict the clinical feasibility of PKA inhibitors in cancer treatment as a novel surrogate marker. The findings demonstrated in this study not only support the involvement of AKIP1 in cancer progression but also indicate the critical role of AKIP1 in determining the effect of PKA signaling on the NF-κB activation cascade. AKIP1 could be used as a molecular determinant for designing tailor-made anti-NF-κB remedy. In AKIP1 high expressing cancer cells, where PKA signaling synergizes with NF-κB activation, the combination of IKK and PKA inhibitors would effectively repress cell growth and induce cancer cell death. In AKIP1-absent cells, activation of PKA cascade by up-regulating the intracellular cAMP level would promote cancer cell apoptosis by antagonizing with the NF-κB activation cascade. We thank Dr. S. Taylor for kindly providing us the anti-AKIP1 antibody.

The Biologic Meaning of the Structural Components of Defibrotide (DF) in the Setting of HIV Disease with Refractory Herpes, Candida, Cryptosporidium Diarrhea and Multi-Organ Failure Against the Background of Down-Regulation of Cellular Immunity, Autocrine Upregulation of Inflammatory Cytokines of TNF-Alpha, IL2, IL1, IL6 and Dysregulation of cAMP/cGMP Protein Kinase A/C Signalling.

Production of DF from native mammalian DNA involves heat controlled depolimerization of polymerized nucleic acids as a function of hyperchromicity measured at wavelengths of 260–280 nm as the ratio of Optical Density (OD) in (disorderly phase)/(OD) in (orderly phase). In double helix DNA this rearrangement can be of the order of 100%. DF is defined to represent a hyperchromicity ratio around 15%. Its structural formula is static: P1-5, (dAp)12–24, (dGp) 10–20, (dTp)13–26, (dCp)10–20. ASH Abstract #4086, 2004 reported sequence identities of the fragments at MW </=560 Da (the calculated MW of alkali Na-salt of ATP), using Reversed Phase- HPLC and (MALDI-TOF) (Oral, Lewis),to represent the capability of DF for 15% molecular rearrangement to include but not limited to: dC, dA, G, dGMP, AMP, dTTP, CTP, ATP, dGTP, CMP, cGMP, dAMP. 4 aptamers were sequenced from the PCR product of the double-stranded Defibrotide precursor (Schroer). We have analyzed Aptamer #4 of the sequence 5′ggtggtggttgtggt against the gag/pol region of HIV, and found 3 homology sites. Translation of the aptamer region in gag is a peptide “PEPTA”, and a pol gene fragment translates the same DNA sequence into TRANS. There is a conserved hairpin in the gag/pol region, just before the aptamer #4 homology. Other homologies of Aptamer #4 include but are not limited to the self-replicating portion of mitochondrial DNA (G.Gorilla, H.Sapiens), Candida tropicalis, but not Herpes, etc. T he reproducibility of these sequences in different batches of DF are still under study, and to be reported at the time of the ASH meeting. DF's mechanism of efficacy as a dose dependent modulator of 2nd messengers, however, are based on a more universally reported data. Highlighted in the literature are the HIV induced loss of bi-directional phosphorilation between Gs GTP/Gs GDP in favor of unidirectional continuous activation of adenylate cyclase (AC) by Gs GTP and secondary uninhibited upregulation of cAMP production. This leads to a known intracellular imbalance between cAMP/cGMP favoring viral replication. Also known are Beta Adrenoreceptor coupling to AC by Gs GTP, and secondary upregulation of autocrine production of inflammatory cytokines of TNF-Alpha, IL6, IL2, IL1, as well as micelle and spore formation of Candida Albicans (Sabie). Similarly, HSV-1 Alpha0 Promoter isknown to contain a putative cAMP responsive element (CRE) via activating transcriptor protein ATP/cAMP CRE binding protein CREB, NF-kB via CA ((Danaher). Reported are upregulation of cAMP, ATP, NADH, gluthathione, 2–3 DPG, NADP /NADPH, and AC by DF, albeit as static events. We will report on an in-vivo dose system aiming at resetting the broken feedback loops of bi-directional phosphorilation reactions as a common denominator for DF's mechanism of efficacy. The patient data dose-stratified from 40mg/kg/day to 300mg/kg/day using either approach are to be detailed at the time of the upcoming ASH meetings.

Multisite phosphorylation of the cAMP response element-binding protein CREB by a diversity of protein kinases

The prevailing view of stimulus-induced activation of the transcription factor cAMP response element-binding protein (CREB) presumes phosphorylation at serine-133. Although, phosphorylation of this residue seems to be necessary, it is not sufficient to trigger CREB-driven transcription, indicating that other phosphoserine-133-independent mechanisms are required for full activation of CREB. One of these additional regulatory mechanisms influencing the transcriptional state of CREB may involve multiple phosphorylation events on other phosphoacceptor sites in the protein. This review focuses on the phosphorylation modifications of CREB by distinct protein kinases and discusses the possible implications for the function of CREB.

CREB activation induced by mitochondrial dysfunction triggers triglyceride accumulation in 3T3-L1 preadipocytes

Several mitochondrial pathologies are characterized by lipid redistribution and microvesicular cell phenotypes resulting from triglyceride accumulation in lipid-metabolizing tissues. However, the molecular mechanisms underlying abnormal fat distribution induced by mitochondrial dysfunction remain poorly understood. In this study, we show that inhibition of respiratory complex III by antimycin A as well as inhibition of mitochondrial protein synthesis trigger the accumulation of triglyceride vesicles in 3T3-L1 fibroblasts. We also show that treatment with antimycin A triggers CREB activation in these cells. To better delineate how mitochondrial dysfunction induces triglyceride accumulation in preadipocytes, we developed a low-density DNA microarray containing 89 probes, which allows gene expression analysis for major effectors and/or markers of adipogenesis. We thus determined gene expression profiles in 3T3-L1 cells incubated with antimycin A and compared the patterns obtained with differentially expressed genes during the course of in vitro adipogenesis induced by a standard pro-adipogenic cocktail. After an 8-day treatment, a set of 39 genes was found to be differentially expressed in cells treated with antimycin A, among them CCAAT/enhancer-binding protein alpha (C/EBPalpha), C/EBP homologous protein-10 (CHOP-10), mitochondrial glycerol-3-phosphate dehydrogenase (GPDmit), and stearoyl-CoA desaturase 1 (SCD1). We also demonstrate that overexpression of two dominant negative mutants of the cAMP-response element-binding protein CREB (K-CREB and M1-CREB) and siRNA transfection, which disrupt the factor activity and expression, respectively, inhibit antimycin-A-induced triglyceride accumulation. Furthermore, CREB knockdown with siRNA also downregulates the expression of several genes that contain cAMP-response element (CRE) sites in their promoter, among them one that is potentially involved in synthesis of triglycerides such as SCD1. These results highlight a new role for CREB in the control of triglyceride metabolism during the adaptative response of preadipocytes to mitochondrial dysfunction.

Signal-dependent Control of Gluconeogenic Key Enzyme Genes through Coactivator-associated Arginine Methyltransferase 1

Together with impaired glucose uptake in skeletal muscle, elevated hepatic gluconeogenesis is largely responsible for the hyperglycemic phenotype in type II diabetic patients. Intracellular glucocorticoid and cyclic adenosine monophosphate (cAMP)/protein kinase A-dependent signaling pathways contribute to aberrant hepatic glucose production through the induction of gluconeogenic enzyme gene expression. Here we show that the coactivator-associated arginine methyltransferase 1 (CARM1) is required for cAMP-mediated activation of rate-limiting gluconeogenic phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) and glucose-6-phosphatase genes. Mutational analysis showed that CARM1 mediates its effect via the cAMP-responsive element within the PEPCK promoter, which is identified here as a CARM1 target in vivo. In hepatocytes, endogenous CARM1 physically interacts with cAMP-responsive element binding factor CREB and is recruited to the PEPCK and glucose-6-phosphatase promoters in a cAMP-dependent manner associated with increased promoter methylation. CARM1 might, therefore, represent a critical component of cAMP-dependent glucose metabolism in the liver.

Induction of intracellular signalling in human endothelial cells by the hyaluronan-binding protease involves two distinct pathways

Recently a novel plasma serine protease with high affinity to hyaluronic acid and glycosaminoglycans, such as heparin and heparan sulfate, has been described and termed hyaluronan-binding protease (HABP). HABP cleaves kininogen in vitro, releasing the vasoactive peptide bradykinin, and activates plasminogen activators, suggesting a vascular cell-directed physiological function of this novel plasma protease. Here we show that HABP stimulates human umbilical vein endothelial cells (HUVECs) by activating two distinct cell-surface receptors. On the one hand, HABP releases bradykinin from cell surface-bound or soluble kininogen and triggers a bradykinin B2-receptor-dependent mobilisation of intracellular Ca2+. On the other hand, HABP activates the p44/42-dependent MAPK (ERK1/2) signalling cascade independent of the B2-receptor, but involving the fibroblast growth factor receptor-1 and basic fibroblast growth factor. This signalling pathway leads to phosphorylation of the kinases Raf, MEK1/2 and ERK1/2. The extracellular activity of HABP also affects the gene expression level through phosphorylation of two transcription factors, the cAMP-responsive element binding protein CREB and the proto-oncogene c-Myc. Our results indicate a proangiogenic potential of HABP, which, in combination with a profibrinolytic activity, directs the physiological function of this plasma protease to processes in which clot lysis, cell motility and neovascularisation are pivotal processes, e.g., in wound healing, tissue repair and tumour progression.

Regulation of gene expression by cyclic GMP.

Cyclic GMP, produced in response to nitric oxide and natriuretic peptides, is a key regulator of vascular smooth muscle cell contractility, growth, and differentiation, and is implicated in opposing the pathophysiology of hypertension, cardiac hypertrophy, atherosclerosis, and vascular injury/restenosis. cGMP regulates gene expression both positively and negatively at transcriptional as well as at posttranscriptional levels. cGMP-regulated transcription factors include the cAMP-response element binding protein CREB, the serum response factor SRF, and the nuclear factor of activated T cells NF/AT. cGMP can regulate CREB directly, through phosphorylation by cGMP-dependent protein kinase, or indirectly, through activation of mitogen-activated protein kinase pathways; regulation of SRF and NF/AT by cGMP is indirect, through modulation of RhoA and calcineurin signaling, respectively. Downregulation of the RNA-binding protein HuR by cGMP leads to destabilization of guanylate cyclase mRNA, but this posttranscriptional mechanism may affect many more cGMP-regulated genes. In this review, we discuss the role of cGMP-regulated gene expression in (patho)physiological processes most relevant to the cardiovascular system, such as regulation of vascular tone, cardiac hypertrophy, phenotypic modulation of vascular smooth muscle cells, and regulation of cell proliferation and apoptosis.

NO activation of fos promoter elements requires nuclear translocation of G-kinase I and CREB phosphorylation but is independent of MAP kinase activation.

We have shown that nitric oxide (NO) regulates c-fos gene expression via cGMP-dependent protein kinase (G-kinase), but NO's precise mechanism of action is unclear. We now demonstrate that: (1) NO targets two transcriptional elements in the fos promoter, i.e., the fos AP-1 binding site and the cAMP-response element (CRE); (2) NO activation of these two enhancer elements requires the CRE binding protein CREB because a dominant negative CREB fully inhibits NO transactivation of reporter genes whereas dominant negative Fos or CCAAT enhancer binding proteins have no effect; (3) CREB is phosphorylated by G-kinase in vitro and its phosphorylation increases in vivo when G-kinase is activated either directly by cGMP or indirectly by NO via soluble guanylate cyclase; (4) NO activation of fos promoter elements requires nuclear translocation of G-kinase but not activation of mitogen-activated protein kinases.

Glucagon-like peptide 1 stimulates insulin gene promoter activity by protein kinase A-independent activation of the rat insulin I gene cAMP response element.

Glucagon-like peptide 1 (GLP-1), a hormonal activator of adenyl cyclase, stimulates insulin gene transcription, an effect mediated by the cAMP response element (CRE) of the rat insulin I gene promoter (RIP1). Here we demonstrate that the signaling mechanism underlying stimulatory effects of GLP-1 on insulin gene transcription results from protein kinase A (PKA)-independent activation of the RIP1 CRE. Although GLP-1 stimulates cAMP production in rat INS-1 insulinoma cells, we find accompanying activation of a -410-bp RIP1 luciferase construct (-410RIP1-LUC) to exist independently of this second messenger. GLP-1 produced a dose-dependent stimulation of -410RIP1-LUC (EC50 0.43 nmol/l), an effect reproduced by the GLP-1 receptor agonist exendin-4 and abolished by the antagonist exendin(9-39). Activation of RIP1 by GLP-1 was not affected by cotransfection with dominant-negative Gs alpha, was not blocked by cAMP antagonist Rp-cAMPS, and was insensitive to PKA antagonist H-89. Truncation of -410RIP1-LUC to generate -307-, -206-, and -166-bp constructs revealed 2 segments of RIP1 targeted by GLP-1. The first segment, not regulated by forskolin, was located between -410 and -307 bp of the promoter. The second segment, regulated by both GLP-1 and forskolin, included the CRE and was located between -206 and -166 bp. Consistent with these observations, stimulatory effects of GLP-1 at RIP1 were reduced after introduction of delta-182 and delta-183/180 inactivating deletions at the CRE. The action of GLP-1 at -410RIP1-LUC was also reduced by cotransfection with A-CREB, a genetically engineered isoform of the CRE binding protein CREB, which dimerizes with and prevents binding of basic-region-leucine-zipper (bZIP) transcription factors to the CRE. In contrast, the action of GLP-1 at the CRE was not blocked by cotransfection with M1-CREB, an isoform that lacks a consensus serine residue serving as substrate for PKA-mediated phosphorylation. On the basis of these studies, it is proposed that PKA-independent stimulatory actions of GLP-1 at RIP1 are mediated by bZIP transcription factors related in structure but not identical to CREB.

Mechanisms of transcriptional activation of cAMP-responsive element-binding protein CREB.

The CREB-CREM transcription factors are the main gene regulatory effectors of the cAMP signaling pathway. The investigations of this family of transcription factors had a profound impact on the understanding of signaling-induced gene transcription. Here we discuss some key aspects of the underlying biology, review transcriptional activation by CREB proteins through transcription cofactors and present novel insights into the context- and position-specific function of CREB on complex genes.

Co-stimulation of human peripheral blood mononuclear cells with IL-2 and anti-CD3 monoclonal antibodies induces phosphorylation of CREB

Phosphorylation of the cAMP-response element binding protein CREB within 1 h of CD2 but not CD3 cross-linking of human PBMC was recently demonstrated. The absence of P-CREB following CD3 cross-linking was unexpected, as other laboratories reported increased phosphorylation of CREB following CD3 cross-linking of the Jurkat lymphocyte cell line. Due to Jurkat T-cells being IL-2-independent, it was postulated that IL-2 might provide a necessary co-stimulus for phosphorylation of CREB in primary lymphocytes. Therefore, P-CREB was evaluated following co-stimulation of human PBMC through the IL-2 and CD2 or CD3 receptors. IL-2 did not further augment phosphorylation of CREB following CD2 cross-linking. However, while neither IL-2 nor CD3 cross-linking alone induced P-CREB, a 4.5-fold increase in phosphorylation of CREB within 1 h of IL-2/CD3 co-stimulation was observed. Phosphorylation was not associated with the induction of cAMP, and inhibition of PKA signaling had no effect on P-CREB. Consistent with signal transduction through p56 lck or p59 fyn, inhibition of PTK signaling reduced phosphorylation 50%. Interestingly, inhibiting PKC signaling with calphostin C further increased P-CREB levels 3-fold over that observed in IL-2/CD3 co-stimulated cells not pretreated with a PKC inhibitor. In contrast to previous studies performed in the absence of exogenous IL-2, no increase in binding of CREB to a 32P-labeled oligonucleotide probe was observed by electrophoretic mobility shift assay. These data suggest that the IL-2 and CD3 signaling pathways provide a necessary and co-operative stimulus promoting phosphorylation of CREB following receptor cross-linking.

Transcriptional regulation of the CYP11A1 and ferredoxin genes

The first step in the synthesis of all steroids is the cleavage of cholesterol side chain, catalyzed by an electron transport system located in mitochondria consisting of ferredoxin reductase, ferredoxin, and cytochrome P450scc. These proteins are present in adrenal, gonad, placenta, and some parts of the brain. In addition, ferredoxin and ferredoxin reductase are also found in the kidney and liver. Whereas ferredoxin reductase levels remain constant in the cell, ferredoxin and P450scc levels are stimulated by trophic hormones using cAMP as an intracellular messenger. The ferredoxin promoter is relatively simple, consisting of a TATA box and two Sp1-binding sites. This simple module is enough to direct cAMP-dependent transcription in a steroidogenic cell-specific fashion. The regulatory region for the P450scc gene is more complex, containing many protein binding sites for different regulation purposes. Its TATA box directs cAMP-dependent transcription in a cell-type-specific manner. A transcription factor, steroidogenic factor 1 (SF1), activates P450scc gene expression. The tissue-specific expression of the P450scc gene is probably accomplished through the interaction of SF1 with other protein factors located further upstream of the control region. SF1 may also be involved in the cAMP response. An upstream region binding to cAMP-Responsive Element Binding Protein CREB and AP1 can respond to cAMP for gene activation. These analyses of regulatory elements provide the structural architecture for transcriptional regulation of the ferredoxin and the CYP11A11 gene.