Geniposide ameliorated fluoxetine-suppressed neurite outgrowth in Neuro2a neuroblastoma cells.

AIM: Fluoxetine (FXT), a selective serotonin reuptake inhibitor (SSRI), is one of the most common psychiatric medications clinically prescribed; while over-produced serotonin may suppress neurite development. The role of major iridoids like geniposide (GPS) and genipin (GNP) from Gardenia jasminoides Ellis fruit (family Rubiaceae) in ameliorating the anti-neurite outgrowth effect of FXT is poorly understood. In this study, the effects of these iridoids on FXT-suppressed neurite outgrowth in Neuro2a neuroblastoma cells were investigated. MAIN METHODS: Neuro2a cells were treated with FXT and GPS. The effect of GPS-FXT co-treatment on neurite outgrowth was observed using inverted phase-contrast microscope imaging system, while neurite outgrowth markers - microtubule-associated protein-2 (MAP2) and growth-associated protein 43 (GAP43) were analyzed using RT-PCR, Western blot and immunofluorescence staining. The transcription factor-cAMP response element binding (CREB), and signaling pathways - mitogen-activated protein kinase (MAPK) and protein kinase B/mammalian target of rapamycin (AKT/mTOR) were also analyzed with the help of Western blot. KEY FINDINGS: The results showed that FXT decreased the neurite outgrowth in Neuro2a cells and also downregulated gene and protein expression of MAP2 and GAP43. It also downregulated the protein expression of phosphorylated-CREB, MAPK, and AKT/mTOR signaling pathways. In contrast, GPS counteracted the effects of FXT. GPS-FXT co-treatment increased the percentage of neurite-bearing cells by 3.6-fold at 200 µM as compared to FXT treatment only. SIGNIFICANCE: This study has provided the possible molecular mechanism showing how FXT exerted its detrimental side-effects on the neurite differentiation, and via the same mechanism how GPS attenuated these side effects.

The protective effect of zeranol in cerebral ischemia reperfusion via p-CREB overexpression.

AIMS: Cerebral ischemia reperfusion (I/R) is a neurovascular disease leading to cerebral damage. It was found that postmenopausal women are liable to more dangerous effects than men at same age in stroke. The objective of this study is to investigate the neuroprotective effect of zeranol against cerebral ischemia reperfusion in ovariectomized rats. MAIN METHODS: 36 female wistar rats divided in to 3 groups: sham group, I/R group (where I/R was induced 7 weeks after ovariectomy), zeranol group (0.5 mg/kg every 3 days for 5 weeks before I/R). Cerebral ischemia reperfusion (I/R) was performed by bilateral common carotid artery occlusion then de-ligated to restore blood flow. After 24 h of reperfusion, rats performed cylinder test to evaluate behavioral dysfunction followed by decapitation. Brain tissues were collected for biochemical measures such as oxidative stress marker malondialdehyde, antioxidant markers reduced glutathione, inflammatory markers (interleukin-1 beta, tumor necrosis factor alpha, and inducible nitric oxide synthase), matrix metalloproteinase-9, adenosine triphosphate, brain derived neurotrophic factor, glucose transporter-3, phosphorylated c-AMP response element binding protein and finally nissl staining for histopathological examination. KEY FINDINGS: The zeranol administered group showed a reversal of neuronal damage caused by ischemia evidenced by the decrease in MDA, IL-1ß, TNF-α, and MMP-9 levels, increase GSH, and ATP levels, decrease expression of iNOS in both regions cortex and hippocampus, increase protein level of p-CREB, GLUT-3 and BDNF, increase number of intact neuron cells in both regions and attenuated histological changes in both cortex and hippocampus regions. SIGNIFICANCE: Zeranol has neuroprotective potential against cerebral ischemia reperfusion in ovariectomized rats.

Luteolin inhibits colorectal cancer cell epithelial-to-mesenchymal transition by suppressing CREB1 expression revealed by comparative proteomics study.

The cyclic AMP (cAMP) response element binding protein 1 (CREB1) is a promising target for cancer therapy. Here, we report that luteolin, a natural product, inhibits the expression of CREB1 at the transcriptional level and blocks epithelial-to-mesenchymal transition (EMT) of colorectal cancer cells. Treatment of colorectal cancer cells with luteolin induced mesenchymal-to-epithelial transition, reduced the expressions of mesenchymal markers and inhibited cell mobility in vitro. Through comparison of the proteomic profile of HCT-116 cells with and without luteolin treatment, we identified 366 differentially expressed proteins. Bioinformatics analysis revealed that downregulation of CREB1 plays a central role in this process. Immunoblot analysis verified that the protein levels of CREB1 and its downstream target genes were decreased in luteolin-treated cells. Moreover, forced expression of CREB1 abolished the inhibitory effect of luteolin on colorectal cancer cells, suggesting the important role of CREB1 in this process. Furthermore, luciferase reporter assay and examination of the half-life of CREB1 following inhibition of new protein synthesis by cycloheximide (CHX) revealed that luteolin inhibits the expression of CREB1 at the transcriptional level. In summary, our results demonstrated that suppressing the expression of CREB1 is crucial in the mechanism-of-action of luteolin inhibiting EMT of colorectal cancer cells. SIGNIFICANCE: It is no doubt that understanding the mechanism-of-action of natural products at the molecular level is important for their translational application. Proteomics is a powerful platform to explore the effects of natural products on the cells. In this study, we compared the proteomic profile of HCT-116 colorectal cancer cells with and without luteolin treatment to investigate the mechanism-of-action of luteolin against colorectal cancer cells. Subsequent bioinformatics analysis revealed that CREB1 could be one of the main targets of luteolin against colorectal cancer cells. Downregulation of CREB1 by luteolin affects glucagon signaling pathway and cAMP signaling pathway. The proteomics findings were verified with mechanistic analyses. We first identified that luteolin decreased the mRNA and protein levels of CREB1 and its downstream target genes. We then found that luteolin inhibits CREB1 expression at the transcriptional level by real-time PCR and luciferase reporter assay which confirmed by examination of the half-life of CREB1 following inhibition of new protein synthesis by cycloheximide (CHX). Finally, we generated CREB1-overexpressing stable cell line and showed that ectopic expression of CREB1 abolished the inhibitory effect of luteolin on colorectal cancer cells and restored the expression levels of CREB1 target genes in colorectal cancer cells, and thereby demonstrated the critical role of CREB1 in the mechanism-of-action of luteolin against colorectal cancer. In summary, we revealed a novel mechanism-of-action of luteolin against colorectal cancer cell by the combination of proteomics discovery and mechanistic analyses.

CCAR2 Is Required for Proliferation and Tumor Maintenance in Human Squamous Cell Carcinoma.

CCAR2 is a widely expressed protein involved in the regulation of a variety of transcriptional complexes. High expression of CCAR2 correlates with poor outcomes in many human tumor types such as squamous cell carcinoma (SCC). Paradoxically, loss of Ccar2 in the mouse results in an increased tumor burden, suggesting that CCAR2 may in fact function as a tumor suppressor. This tumor suppressor function is dependent on p53, a protein that is inactivated in the vast majority of SCC tumors, leaving the role of CCAR2 in p53-null tumors unclear. We sought to identify p53-independent CCAR2 functions in SCC and to examine its role in tumorigenesis. We found that CCAR2 is highly overexpressed in p53-deficient SCC cell lines compared with normal primary keratinocytes due to increased protein stability. We identify a role for CCAR2 in promoting the stability of the transcription factors RFX1 and CREB1, which are both required for proliferation. Finally, we show that CCAR2 is required for proliferation in vitro and in established SCC tumors in vivo. Our data suggest an important role for CCAR2 in maintaining cell cycle progression and promoting SCC tumorigenesis.

Regulation of cyclic adenosine monophosphate response element binding protein on renin expression in kidney via complex cyclic adenosine monophosphate response element-binding-protein-binding protein/P300 recruitment.

INTRODUCTION: Renin synthesis and release is the rate-limiting step in the renin-angiotensin system, because cyclic adenosine monophosphate (cAMP) has been identified as dominant pathway for renin gene expression, and cAMP response element-binding protein (CREB) is found in the human and mouse renin promoter. This study aimed to evaluate the role of CREB in expression of the renin gene. MATERIALS AND METHODS: We created conditional deletion of CREB in mice with low-sodium diet, specifically in renin cells of the kidney. To assess the effect of CREB on renin expression, immunostaining of renin was used in samples from wild-type mice and mice with gene knock-down of CREB. Cyclic AMP response element-binding-protein-binding protein (CBP) and p300 were measured in cultured renin cells of the mice, and RNA detection was done with real-time polymerase chain reaction. RESULTS: With low-sodium diet, renin was expressed along the whole wall of the afferent glomerular arterioles in wild-type mice, while there was no increase or even decrease in renin expression in CREB-specific deletion mice; RNA level of renin in cultured cells decreased by 50% with single knock-down of CREB, CBP, or p300, and decreased 70% with triple knock-down of CREB, CBP, and p300. CONCLUSIONS: This study found that CREB was important for renin synthesis and the role of CREB can be achieved through the recruitment of co-activators CBP and p300.

Prognostic value of coexistence of abnormal expression of micro-RNA-200b and cyclic adenosine monophosphate-responsive element-binding protein 1 in human astrocytoma.

Our aim was to investigate the expression of micro-RNA-200b (miR-200b) and cAMP-responsive element-binding protein 1 (CREB-1) in astrocytoma and its efficacy for predicting outcome. Both miR-200b and CREB-1 messenger RNA expression was measured in 122 astrocytomas and 30 nonneoplastic brain specimens by quantitative real-time polymerase chain reaction. Expression of miR-200b was significantly lower in astrocytoma than in nonneoplastic brain (P < .001), whereas CREB-1 messenger RNA expression was significantly elevated in the tumors (P < .001). Both miR-200b down-regulation and CREB-1 up-regulation were significantly associated with advanced pathologic grade (P = .002 and P = .006, respectively). Low miR-200b expression correlated negatively with Karnofsky performance score (P = .03), and high CREB-1 expression correlated positively with mean tumor diameter (P = .03). By Kaplan-Meier analysis, low miR-200b, high CREB-1, and coexistence of abnormal miR-200b and CREB-1 expression (low miR-200b/high CREB-1) were predictive of shorter progression-free survival and overall survival in both grade III and grade IV astrocytoma. By multivariate analysis, only low miR-200b/high CREB-1 expression was an independent prognostic factor for poor prognosis in astrocytoma of advanced grade. Both miR-200b and CREB-1 may play important cooperative roles in the progression of human astrocytoma. The efficacy of miR-200b and CREB-1 together as a predictor of prognosis in astrocytoma patients is shown for the first time.

Detection of CREB phosphorylation via Zr (IV) ion mediated signal amplification.

Phosphorylation of protein plays a vital regulatory role in a variety of biological processes. We herein report a novel method to assay the level of phosphorylated cAMP-response element binding protein (CREB) via Zr(4+) mediated signal amplification using gold nanoparticle/DNA/methylene blue (GNP/DNA/MB) nanocomposites. In this method the probe DNA immobilized at a gold electrode surface can specifically and efficiently recognize the phosphorylated target protein CREB. Then Zr(4+) links the phosphorylated CREB with GNP/DNA/MB nanocomposites by coordinating the phosphate groups on both CREB and the nanocomposites. Since the nanocomposites can provide high sensitivity (limit of detection: 0.25 nM) for the detection, efficient and highly sensitive bioanalysis of the expression level of phosphorylated protein CREB in human placenta tissues has also been conducted in this work. Our method is reported which shows acceptable stability, reproducibility for assaying of the protein phosphorylation states in real biosamples under physiological and pathological conditions with great potential for clinical applications in future.

Neuroprotective effect of estradiol-loaded poly(lactic-co-glycolic acid) nanoparticles on glutamate-induced excitotoxic neuronal death.

Different concentrations of estradiol (E2)-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (E2-PLGA-NPs) were synthesized using the emulsion-diffusion method. Transmission electron microscopy results showed that the average particle size of E2-PLGA-NPs was 98 ± 1.9 nm when stabilized with polyvinyl alcohol and 103 ± 4.9 nm when stabilized with Tween-80. Fourier transform-infrared spectroscopy with diamond attenuated total reflectance was used to identify the presence or absence of E2 molecules in PLGA nanocapsules. Cell proliferation was assessed after treating SH-SY5Y neuroblastoma cells with 1 nM-1 µM of E2 and E2-PLGA-NPs. The neuroprotective efficacy against glutamate-induced excitotoxicity was also investigated in SH-SY5Y neuroblastoma cells. Neuroprotection was greater in E2-PLGA-NP-treated cells than in cells treated with the same concentration of E2. Furthermore, E2- and E2-PLGA-NP-treated cells expressed more p-ERK1/2 and p-CREB than cells treated with glutamate only. Moreover, the expression of p-ERK1/2 was higher than that of p-CREB. In this study, p-ERK1/2 had a greater influence on the neuroprotective effect of E2 and E2-PLGA-NPs than p-CREB.

Grape powder supplementation prevents oxidative stress-induced anxiety-like behavior, memory impairment, and high blood pressure in rats.

We examined whether or not grape powder treatment ameliorates oxidative stress-induced anxiety-like behavior, memory impairment, and hypertension in rats. Oxidative stress in Sprague-Dawley rats was produced by using L-buthionine-(S,R)-sulfoximine (BSO). Four groups of rats were used: 1) control (C; injected with vehicle and provided with tap water), 2) grape powder-treated (GP; injected with vehicle and provided for 3 wk with 15 g/L grape powder dissolved in tap water), 3) BSO-treated [injected with BSO (300 mg/kg body weight), i.p. for 7 d and provided with tap water], and 4) BSO plus grape powder-treated (GP+BSO; injected with BSO and provided with grape powder-treated tap water). Anxiety-like behavior was significantly greater in BSO rats compared with C or GP rats (P < 0.05). Grape powder attenuated BSO-induced anxiety-like behavior in GP+BSO rats. BSO rats made significantly more errors in both short- and long-term memory tests compared with C or GP rats (P < 0.05), which was prevented in GP+BSO rats. Systolic and diastolic blood pressure was significantly greater in BSO rats compared with C or GP rats (P < 0.05), whereas grape powder prevented high blood pressure in GP+BSO rats. Furthermore, brain extracellular signal-regulated kinase-1/2 (ERK-1/2) was activated (P < 0.05), whereas levels of glyoxalase-1 (GLO-1), glutathione reductase-1 (GSR-1), calcium/calmodulin-dependent protein kinase type IV (CAMK-IV), cAMP response element-binding protein (CREB), and brain-derived neurotrophic factor (BDNF) were significantly less (P < 0.05) in BSO but not in GP+BSO rats compared with C or GP rats. We suggest that by regulating brain ERK-1/2, GLO-1, GSR-1, CAMK-IV, CREB, and BDNF levels, grape powder prevents oxidative stress-induced anxiety, memory impairment, and hypertension in rats.

Multiplexed analysis of proteins in tissue using multispectral fluorescence imaging.

We present a new application of multispectral analysis for subcellular measurement of multiple proteins in formalin-fixed paraffin embedded tissue and cells. Typically, the targets of interest are present in the same or spatially overlapping cellular compartments. Such co-localization can complicate analysis and interpretation of the images obtained using traditional fluorescence, especially when spectrally overlapping labels are present. The spectral properties of currently available fluorescent dyes set an upper limit to the number of molecules that can be detected simultaneously with traditional fluorescence. By exciting a set of fluorophores at the same wavelength and unmixing their emission signals from background autofluorescence, we were able to image three targets in a single channel. This parallel imaging approach provides significant advantages for multiplexed analysis of tissues and cells.

[Expression of CREB/Bcl-2 in bone marrow mononuclear cells of children with acute leukemia].

OBJECTIVE: To study the expression and role of cyclic-AMP response binding protein (CREB) and Bcl-2 in children with acute leukemia. METHODS: Ninety-two children with acute leukemia (leukemia group) and 30 children with non-hematologic malignancies (control group) were enrolled. The mRNA and protein expression of CREB and Bcl-2 in bone marrow mononuclear cells were measured by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot. RESULTS: The mRNA and protein expression of CREB and Bcl-2 in the leukemia group was significantly higher than that in the control group (p<0.01). There were no significant differences in the expression of CREB and Bcl-2 between acute lymphoblastic leukemia and acute myeloid leukemia subgroups. At the initial diagnosis, the mRNA and protein expression of CREB and Bcl-2 in children with extramedullary infiltration was higher than that in children without (p<0.05). In the leukemia group, the mRNA and protein expression of CREB and Bcl-2 in the complete remission subgroup was significantly lower than that in the non-complete remission subgroup (p<0.01). High mRNA expression of CREB and Bcl-2 in the leukemia group was positively correlated with peripheral blood leucocyte counts (r=0.62, 0.71 respectively, p<0.05). There was a positive correlation between mRNA and protein expression of CREB and Bcl-2 (r=0.75, 0.68 respectively; p<0.05). CONCLUSIONS: The expression of CREB and Bcl-2 may be correlated with the pathogenesis and clinical prognosis of childhood leukemia, however, their expression may not be associated with the classification of acute leukemia.

Altered levels of extracellular signal-regulated kinase signaling proteins in postmortem frontal cortex of individuals with mood disorders and schizophrenia.

BACKGROUND: The extracellular-regulated protein kinase (ERK) pathway has been implicated in processes such as neuronal plasticity and resilience in psychiatric disorders including major depressive disorder (MDD), bipolar disorder (BPD), and schizophrenia. The extent of the possible involvement of this pathway in psychiatric disorders remains unknown, as does its potential utility as a pharmacological target for the future development of novel therapeutics. METHODS: Western blot analyses were used to measure levels of different proteins-Rap1, B-Raf, MEK1, MEK2, ERK1/2, RSK1, CREB, NSE, and beta-actin-in the postmortem frontal cortex of individuals with schizophrenia, MDD, and BPD, as well as healthy non-psychiatric controls. RESULTS: Levels of most studied protein members of the ERK cascade were lower in individuals with psychiatric disorders than controls; differences between psychiatric groups were not statistically significant. In general, protein levels were lower in individuals with schizophrenia than in those with BPD or MDD, but protein levels varied across groups. LIMITATIONS: The small number of individuals in each diagnostic group may limit our interpretation of the results. Factors such as postmortem interval, medication status at time of death, and mood state at time of death may also have influenced the findings. DISCUSSION: The results are consistent with the hypothesis that the ERK pathway is implicated in reduced neuronal plasticity associated with the course of these psychiatric illnesses. The results warrant an expanded investigation into the activity of other members of this pathway as well as other brain areas of interest.

Effects of 4-week treatment with lithium and olanzapine on levels of brain-derived neurotrophic factor, B-cell CLL/lymphoma 2 and phosphorylated cyclic adenosine monophosphate response element-binding protein in the sub-regions of the hippocampus.

A large body of evidence indicates that lithium, the prototype mood stabilizer in the treatment of bipolar disorder, has diverse neuroprotective and neurotrophic actions, and the actions are associated with its efficacy in treating bipolar disorder. It has been suggested that up-regulation of neurotrophic and neuroprotective factors including brain-derived neurotrophic factor (BDNF) and B-cell CLL/lymphoma 2 (Bcl-2) may underlie these neuroplastic actions of the drug. Olanzapine, an atypical anti-psychotic drug, has been shown to be an effective mood stabilizer. Olanzapine also has neurotrophic and neuroprotective actions, and these actions may underlie the efficacy of the drug for bipolar disorder and schizophrenia. However, the molecular mechanism by which the drug produces the neuroplastic actions is poorly understood. To understand a common molecular mechanism underlying the neuroplastic actions of lithium and olanzapine, we assessed the effect of 4-week lithium and olanzapine treatment on the levels of BDNF, Bcl-2 and cyclic adenosine monophosphate response element-binding protein (CREB), a transcription factor involved in expression of BDNF and Bcl-2, in the dentate gyrus and hippocampal area CA1. Our results show that 4-week treatment with both olanzapine and lithium increases the levels of Bcl-2 and CREB in the dentate gyrus and hippocampal area CA1. Four-week lithium treatment up-regulates BDNF in the dentate gyrus, and 4-week olanzapine treatment marginally did so. Neither drug altered BDNF levels in area CA1. These results suggest that the up-regulation of Bcl-2 and CREB may underlie the neuroplastic actions of olanzapine and lithium.

beta-Carotene promotes the development of NNK-induced small airway-derived lung adenocarcinoma.

AIM: beta-Carotene has shown cancer-preventive effects in preclinical studies while increasing lung cancer mortality in clinical trials. We have shown that beta-carotene stimulates cAMP signalling in vitro. Here, we have tested the hypothesis that beta-carotene promotes the development of pulmonary adenocarcinoma (PAC) in vivo via cAMP signalling. METHODS: PAC was induced in hamsters with the carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), followed by beta-carotene for 1.5 years. Incidence, multiplicity and size of lung tumours were recorded, and phosphorylated CREB and ERK1/2 in tumour cells were determined by Western blots. Cyclic AMP in blood cells was analysed by immunoassays, retinoids in serum and lungs by HPLC. RESULTS: beta-Carotene increased lung tumour multiplicity, lung tumour size, blood cell cAMP, serum and lung levels of retinoids and induced p-CREB and p-ERK1/2 in lung tumours. CONCLUSIONS: Our data suggest that beta-carotene promotes the development of PAC via increased cAMP signalling.

Cooperative regulation of endogenous cAMP-response element binding protein and CCAAT/enhancer-binding protein beta in GH-stimulated c-fos expression.

GH activates the c-fos promoter by regulating multiple transcription factors. This study adds to our understanding of GH-regulated transcription by demonstrating that GH regulates the c-fos cAMP-response element (CRE) and its binding protein, CREB. Activation of the c-fos promoter by GH is impaired by expression of dominant-negative A-CREB. GH stimulates rapid and transient phosphorylation of CREB at Ser 133 (P-CREB), a critical site for transactivation by CREB, in 3T3-F442A preadipocytes. Mutation of this residue impairs GH-induced c-fos expression, suggesting that phosphorylation of CREB at Ser 133 contributes to GH-induced c-fos activation. The MEK inhibitor UO126 impaired the phosphorylation of CREB and that of C/EBPbeta, suggesting that ERKs mediate the phosphorylation of both proteins. UO126, but not the protein kinase A inhibitor H89, blocked GH-induced c-fos mRNA expression. A combination of CREB and C/EBPbeta enhanced c-fos promoter activation, and mutation of the CRE impaired the enhancement, as well as GH-stimulated c-fos activation. GH treatment increased the occupancy of both endogenous phospho-CREB and phospho-C/EBPbeta on the c-fos promoter. The increases were impaired by UO126. The active P-CREB and P-C/EBPbeta are induced by GH to occupy the same c-fos promoter DNA, suggesting that they may participate in a GH-regulated complex on c-fos. These findings suggest that coordinated phosphorylation of CREB and C/EBPbeta in response to GH is mediated by ERK1/2, and that the phosphorylated proteins are part of a regulatory complex that occupies c-fos in vivo to regulate c-fos transcription cooperatively in response to GH.

Regulation of Cox-2 by cyclic AMP response element binding protein in prostate cancer: potential role for nexrutine.

We recently showed that Nexrutine, a Phellodendron amurense bark extract, suppresses proliferation of prostate cancer cell lines and tumor development in the transgenic adenocarcinoma of mouse prostate (TRAMP) model. Our data also indicate that the anti-proliferative effects of Nexrutine are emediated in part by Akt and Cyclic AMP response element binding protein (CREB). Cyclooxygenase (Cox-2), a pro-inflammatory mediator, is a CREB target that induces prostaglandin E(2) (PGE(2)) and suppresses apoptosis. Treatment of LNCaP cells with Nexrutine reduced tumor necrosis factor alpha-induced enzymatic as well as promoter activities of Cox-2. Nexrutine also reduced the expression and promoter activity of Cox-2 in PC-3 cells that express high constitutive levels of Cox-2. Deletion analysis coupled with mutational analysis of the Cox-2 promoter identified CRE as being sufficient for mediating Nexrutine response. Immunohistochemical analysis of human prostate tumors show increased expression of CREB and DNA binding activity in high-grade tumors (three-fold higher in human prostate tumors compared to normal prostate; P = .01). We have identified CREB-mediated activation of Cox-2 as a potential signaling pathway in prostate cancer which can be blocked with a nontoxic, cost-effective dietary supplement like Nexrutine, demonstrating a prospective for development of Nexrutine for prostate cancer management.

cAMP-responsive element-binding protein expression and regulation in the mouse preimplantation embryo.

Gene expression from the new embryonic genome is required for normal preimplantation embryo development. Two members of the cAMP-responsive element-binding protein (Creb) family of transcription factors, Creb1 and activating transcription factor 1 (Atf1), are essential for normal preimplantation development. These transcription factors are activated by phosphorylation. Creb1 mRNA was expressed throughout the preimplantation phase. Cytoplasmic immunolocalization of Creb1 was detected in all preimplantation embryo stages. The antigen was largely excluded from the pronuclei/nuclei at embryonic stages except in the mid-cycle two-cell and compacted eight-cell embryo. Activation-state-specific antibodies showed serine 133 phosphorylated Creb1 localization was similar to Creb1 staining, except that there was no increase in staining at the eight-cell stage. Increased staining of phosphorylated Creb1 was observed in the nucleus of mid-cycle two-cell embryos. Increased expression of phosphorylated Creb1 in the two-cell embryo was induced by brief exposure of embryos to ionomycin, but not by a dibutyryl cAMP. This was blocked by buffering intracellular calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester), but not by a cAMP antagonist, Rp-cyclic 3',5'-hydrogen phosphorothioate adenosine. Calmodulin is an intracellular receptor for calcium. Calmodulin mRNA was expressed throughout the preimplantation phase of development. The calmodulin antagonist, W-7, inhibited the ionomycin-induced localization of phosphorylated Creb1 in the nucleus. Treatment of embryos with W-7 caused a dose-dependent inhibition of normal development of zygotes to the blastocysts stage. The study shows Creb1 expression and nuclear localization was dynamically regulated in the early embryo. The marked nuclear accumulation and phosphorylation of Creb1 at the two-cell stage occurred at the time of transcription from the embryonic genome and was regulated in a calcium- and calmodulin-dependent manner.

Variable p-CREB expression depicts different asthma phenotypes.

BACKGROUND: Chromatin modification may play a role in inflammatory gene regulation in asthma. Cyclic adenosine mono-phosphate response element-binding protein (CREB), with the specific co-activator, the CREB-binding protein (CBP), contributes to the acetylation of chromatin and to the transcription of pro-inflammatory genes. OBJECTIVES: To evaluate the expression of CBP and of phospho-CREB (p-CREB) in bronchial biopsies and in peripheral blood mononuclear cells (PBMC) of controls (C), untreated (UA), inhaled steroid treated (ICS) and steroid-dependent asthmatic (SDA) patients. METHODS: We used immunohistochemistry in bronchial biopsies and western blot analysis and immunocytochemistry in PBMC. RESULTS: Cyclic adenosine mono-phosphate response element-binding protein expression, in the epithelium was similar in all groups, while p-CREB expression was increased in UA and in SDA in comparison with ICS and C subjects (C vs UA P = 0.002, C vs SDA P = 0.007), (ICS vs SDA P = 0.005), (ICS vs UA P = 0.001). Interestingly, also in the submucosa, p-CREB was increased in UA and SDA in comparison with ICS and C subjects (C vs UA P = 0.0004) (C vs SDA P < 0.0001) (ICS vs UA P = 0.002) (ICS vs SDA P < 0.0001) and positively correlated with leukocyte infiltration within the bronchi (CD45RB+ cells). Similar results were obtained with PBMC isolated from the same patient groups. Incubation of PBMC in vitro, with fluticasone propionate, decreased the p-CREB expression induced by cytokine activation (interferon-gamma, tumor necrosis factor-alpha). CONCLUSIONS: This study demonstrates that the expression of p-CREB is related, in asthma, to the persistent inflammation according to the disease severity. p-CREB expression can be modulated by glucocorticoids in responsive patients.

Autocrine expression of osteopontin contributes to PDGF-mediated arterial smooth muscle cell migration.

OBJECTIVE: Migration of smooth muscle cells (SMCs) from the media to the intima of arteries is involved in intimal thickening. The platelet-derived growth factor (PDGF) BB is recognized as a major migratory factor for arterial SMCs both in vitro and during neointima formation. Since PDGF acts in synergy with the matrix protein osteopontin (OPN) and also induces its expression, the present study was conceived to explore the role of the OPN produced in an autocrine fashion by PDGF-stimulated SMCs in the migration process and to define regulatory mechanisms of OPN expression. METHODS AND RESULTS: PDGF stimulation of quiescent rat aortic SMCs induced their migration (transfilter assays) and the increase of OPN expression (mRNA and protein assays). Blockade of either OPN expression by a specific short interference RNA (siRNA) or of its function by a blocking antibody decreased the PDGF-stimulated migration by about 70%, demonstrating that autocrine production and excretion of OPN are integral to the PDGF-induced SMC migration. In parallel, SMC stimulation by PDGF also activated the transcription factor CREB essentially through mitogen-activated protein kinase (MAPK) 1/2 and protein kinase A (PKA) pathways. Inhibition of either CREB expression (via siRNA) or function (via dominant-negative CREB) decreased both PDGF-induced SMC migration and OPN expression. SMC transfection with OPN promoter reporter constructs demonstrated that PDGF-induced OPN transcription is mediated by CREB binding to two functional sites of the OPN promoter: a CRE site located at -1403 and an AP-1 site located at -76. CONCLUSION: The present study demonstrates that the autocrine expression of OPN plays a major role in PDGF-induced SMC migration. It further shows that the transcription factor CREB, activated in PDGF-stimulated SMCs, plays a key role in PDGF-induced SMC migration, probably by regulating OPN expression.

A novel cyclic adenosine monophosphate responsive luciferase reporter incorporating a nonpalindromic cyclic adenosine monophosphate response element provides optimal performance for use in G protein coupled receptor drug discovery efforts.

The authors report the characterization of a novel cyclic adenosine monophosphate (cAMP)-responsive luciferase (Luc) reporter that exhibits optimal performance in high-throughput screens of agonist binding at G protein-coupled receptors (GPCRs). This reporter (RIP1-CRE-Luc) incorporates a nonpalindromic cAMP response element (CRE) originally identified within the 5' promoter of the rat insulin 1 gene (RIP1). When multimerized and fused to the coding sequence of firefly luciferase, the CRE of RIP1 allows for the efficient activation of luciferase expression by cAMP-elevating agents or by cAMP itself. Of primary importance is the demonstration that RIP1-CRE-Luc does not exhibit the relatively high levels of basal luciferase activity inherent to reporters incorporating the palindromic CRE first identified in the somatostatin gene promoter. Furthermore, studies of HEK cells expressing class II GPCRs for the cAMP-elevating hormones GLP-1, GIP, and glucagon demonstrate that RIP1-CRE-Luc affords a much wider dynamic range of activation upon exposure to agonist. Such properties of RIP1-CRE-Luc indicate its usefulness as a new and powerful tool for the identification of small-molecule compounds with receptor-stimulating actions or for the identification of constitutively active orphan receptors with cAMP-signaling properties.