Expression Of cAMP Response Element-binding Protein Research Articles

Protection against Morphine-Induced Inhibitory Avoidance Memory Impairment in Rat by Curcumin: Possible Role of Nitric Oxide/ cAMP-Response Element Binding Protein Pathway.

Although a substantial body of research suggests curcumin (CUR) has the preventive potential in memory impairment, the mechanism by which CUR prevents memory loss is still being investigated. This study employs an inhibitory avoidance (IA) model to investigate whether CUR can prevent morphine (Mor)-induced memory impairment as well as the possible role of cAMP-response element binding (CREB) protein, and nitric oxide (NO) signaling in this mechanism. This experimental study was conducted at the Animal Lab of the Physiology Research Center, Kashan University of Medical Sciences (Kashan, Iran) in 2018. Forty rats were randomly divided into four groups: control, CUR (pretreatment gavage of CUR [10 mg/Kg] for 35 days), Mor (7.5 mg/Kg, i.p.), and CUR+Mor (n=10 per group). Following the evaluation of the IA memory and locomotor activity of the animals, the CREB protein expression in the hippocampus and NO metabolites (NOx) level in the brain tissue were also investigated. The data were analyzed using Sigmaplot software (version 14.0) by using the ANOVA, Kruskal-Wallis, Holm-Sidak, and Dunn's post hoc tests. P<0.05 was considered to be statistically significant. In the Mor group, the IA memory of the rats was significantly impaired (P=0.001). CUR prevented the Mor-induced IA memory impairment (P=0.075). While the Mor treatment decreased the phosphorylated CREB (p-CREB) expression, the CUR+Mor cotreatment increased p-CREB expression (P=0.010). Nevertheless, the Mor treatment increased the total CREB expression (P=0.010). The NOx concentration in the brain tissue was decreased following the Mor treatment (P=0.500) but increased after the CUR+Mor cotreatment (P=0.001). The present findings suggest that CUR prevents the memory impairment of rats, possibly through NO and its downstream CREB signaling.

Nerve injury induces transient locus coeruleus activation over time: role of the locus coeruleus-dorsal reticular nucleus pathway.

The transition from acute to chronic pain results in maladaptive brain remodeling, as characterized by sensorial hypersensitivity and the ensuing appearance of emotional disorders. Using the chronic constriction injury of the sciatic nerve as a model of neuropathic pain in male Sprague-Dawley rats, we identified time-dependent plasticity of locus coeruleus (LC) neurons related to the site of injury, ipsilateral (LCipsi) or contralateral (LCcontra) to the lesion, hypothesizing that the LC→dorsal reticular nucleus (DRt) pathway is involved in the pathological nociception associated with chronic pain. LCipsi inactivation with lidocaine increased cold allodynia 2 days after nerve injury but not later. However, similar blockade of LCcontra reduced cold allodynia 7 and 30 days after inducing neuropathy but not earlier. Furthermore, lidocaine blockade of the LCipsi or LCcontra reversed pain-induced depression 30 days after neuropathy. Long-term pain enhances phosphorylated cAMP-response element binding protein expression in the DRtcontra but not in the DRtipsi. Moreover, inactivation of the LCcontra→DRtcontra pathway using dual viral-mediated gene transfer of designer receptor exclusively activated by designer drugs produced consistent analgesia in evoked and spontaneous pain 30 days postinjury. This analgesia was similar to that produced by spinal activation of α2-adrenoreceptors. Furthermore, chemogenetic inactivation of the LCcontra→DRtcontra pathway induced depressive-like behaviour in naïve animals, but it did not modify long-term pain-induced depression. Overall, nerve damage activates the LCipsi, which temporally dampens the neuropathic phenotype. However, the ensuing activation of a LCcontra→DRtcontra facilitatory pain projection contributes to chronic pain, whereas global bilateral LC activation contributes to associated depressive-like phenotype.

LncRNA Eif4g2 improves palmitate-induced dysfunction of mouse β-cells via modulation of Nrf2 activation

Chronic oxidative stress resulting from hyperlipidemia is thought to be a key pathogenic driver of pancreatic β-cell dysfunction in leading to the onset of type 2 diabetes mellitus (T2DM). Long non-coding RNAs (lncRNAs) have been increasingly recognized to regulate dysfunction within pancreatic β-cells in the context of T2DM. In the present study, we sought to comprehensively analyze the roles of lncRNAs in dysfunctional β-cells and mouse islets. Analyses of INS-1E cells were performed by RNA-seq and qRT-PCR after treating with or without 0.5 mM palmitate for 4 days, leading us to identify the novel lncRNA Eif4g2 (lncEif4g2) as a functional regulator within these cells. When we overexpressed lncEif4g2 in INS-1E β-cells and mouse islets, this was sufficient for the reversal of palmitate-mediated reductions in cell viability, insulin production, ATP production by mitochondria, and creation of intracellular reactive oxygen species (ROS) and the dysfunction of mouse islets, with nuclear factor erythroid 2 related factor 2 (Nrf2) activation also being observed. In contrast, when lncEif4g2 was knocked down this led INS-1E cells and mouse islets to become more sensitive to palmitate-induced dysfunction, with reduced Nrf2 nuclear translocation also being detected. When antioxidants were used to treat INS-1E cells and mouse islets, however, these negative effects were reversed. Additional functional analyses revealed lncEif4g2 to be capable of directly binding to miR-3074–5p in β-cells, with the expression of lncEif4g2 and miR-3074–5p being negatively correlated with one another. We further found that cAMP-responsive element binding-protein (CREB) was a miR-3074–5p target gene in these cells, thus at least in part serving as a functional mediator of the lncEif4g2/miR-3074–5p axis within dysfunctional β-cells. In summary, our results thus reveal that lncEif4g2 is able to indirectly regulate the expression of CREB via targeting miR-3074–5p in INS-1E cells and mouse islets, thereby leading to enhanced Nrf2 activation.

Melatonin improves memory defects in a mouse model of multiple sclerosis by up-regulating cAMP-response element-binding protein and synapse-associated proteins in the prefrontal cortex.

Multiple sclerosis is a progressive autoimmune disorder of the myelin sheath and is the most common inflammatory disease of young adults. Up to 65% of multiple sclerosis patients have cognitive impairments such as memory loss and difficulty in understanding and maintaining attention and concentration. Many pharmacological interventions have been used to reverse motor impairments in multiple sclerosis patients; however, none of these drugs improve cognitive function. Melatonin can diffuse through the blood-brain barrier and has well-known antioxidant and anti-inflammatory properties with almost no side effects; it is, therefore, a promising neuroprotective supplement for many neurological diseases, such as multiple sclerosis, Alzheimer's disease, Parkinson's disease, ischemic stroke, and fibromyalgia. However, only some researches have assessed the effect of melatonin on cognitive dysfunction in multiple sclerosis. Here, we evaluated the effects of melatonin supplementation on memory defects induced by cuprizone in a mouse model of multiple sclerosis. Cuprizone (400 mg/kg) and melatonin (80 mg/kg) were administered to SWR/J mice daily for 5 weeks. Open field, tail-flick, and novel object recognition behavioral tests were performed. Also, expression of cAMP-response element-binding protein, synaptophysin, and postsynaptic density protein 95 were measured in the prefrontal cortex. Melatonin significantly improved the memory defects induced by cuprizone toxicity by up-regulating cAMP-response element-binding protein and by increasing expression of the synapse-associated synaptophysin and postsynaptic density protein 95 genes in the prefrontal cortex. These results indicate that melatonin may provide protective effects against memory impairments associated with multiple sclerosis.

CAMP/Protein Kinase A Signaling Inhibits Dlx5 Expression via Activation of CREB and Subsequent C/EBPβ Induction in 3T3-L1 Preadipocytes.

Distal-less homeobox 5 (Dlx5) is a negative regulator of adipogenesis. Dlx5 expression is decreased by adipogenic stimuli, but the mechanisms of Dlx5 downregulation by adipogenic stimuli have not yet been determined. Here, we tested the impact of cAMP/PKA (protein kinase A) signaling induced by 3-isobutyl-1 methyl xanthine (IBMX), forskolin, and 8-CPT-cAMP on the expression of Dlx5 in 3T3-L1 preadipocytes. Significant downregulation of Dlx5 mRNA expression and protein production levels were observed via cAMP/PKA-dependent signaling. Forced expression of cAMP-responsive element-binding protein (CREB) and CCAAT/enhancer-binding protein β (C/EBPβ) was sufficient for downregulation of Dlx5 expression and revealed that CREB functions upstream of C/EBPβ. In addition, C/EBPβ knockdown by siRNA rescued Dlx5 expression in IBMX-treated 3T3-L1 preadipocytes. Luciferase assays using a Dlx5-luc-2935 reporter construct demonstrated the requirement of the Dlx5 promoter region, ranging from −774 to −95 bp that contains two putative C/EBPβ binding elements (site-1: −517 to −510 bp and site-2: −164 to −157 bp), in the suppression of Dlx5 transcription. Consequently, chromatin immunoprecipitation analysis confirmed the importance of site-1, but not site-2, in C/EBPβ binding and transcriptional suppression of Dlx5. In conclusion, we elucidated the underling mechanism of Dlx5 downregulation in IBMX-induced adipogenesis. IBMX activated cAMP/PKA/CREB signaling and subsequently upregulated C/EBPβ, which binds to the Dlx5 promoter to suppress Dlx5 transcription.

Melatonin ameliorates cognitive memory by regulation of cAMP-response element-binding protein expression and the anti-inflammatory response in a rat model of post-traumatic stress disorder

BackgroundPost-traumatic stress disorder (PTSD) is an important psychological disease that can develop following the physical experience or witnessing of traumatic events. The psychopathological response to traumatic stressors increases inflammation in the hippocampus and induces memory deficits. Melatonin (MTG) plays critical roles in circadian rhythm disorders, Alzheimer’s disease, and other neurological disorders. However, the cognitive efficiency of MTG and its mechanisms of action in the treatment of PTSD remain unclear. Thus, the present study investigated the effects of MTG on spatial cognitive impairments stimulated by single prolonged stress (SPS) in rats, an animal model of PTSD. Male rats received intraperitoneal (i.p.) administration of various doses of MTG for 21 consecutive days after the SPS procedure.ResultsSPS-stimulated cognitive impairments in the object recognition task and Morris water maze were reversed by MTG treatment (25 mg/kg, i.p). Additionally, MTG significantly increased cognitive memory-related decreases in cAMP-response element-binding (CREB) protein and mRNA levels in the hippocampus. Our results also demonstrate that MTG significantly inhibited SPS-stimulated cognitive memory impairments by inhibiting the expression of proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in the rat brain.ConclusionThe present results indicate that MTG can be beneficial for SPS-stimulated memory impairments via changes in CREB expression and proinflammatory mediators. Thus, MTG may be a prophylactic strategy for the prevention or mitigation of the progression of some features of the PTSD pathology.

Apocynum venetum Leaf Extract Exerts Antidepressant-Like Effects and Inhibits Hippocampal and Cortical Apoptosis of Rats Exposed to Chronic Unpredictable Mild Stress.

We investigated the effects of Apocynum venetum leaf extract (AVLE) on depressive behaviors and neuronal apoptosis in a chronic unpredictable mild stress (CUMS) rat model of depression. Rats were randomly divided into six groups: control, chronic unpredictable mild stress, fluoxetine, AVLE30, AVLE60, and AVLE120. Except for the control group, all rats were submitted to chronic unpredictable mild stress paradigms for four weeks to induce depressive behavior. Neuronal apoptosis was assessed by the terminal deoxynucleotidyl transferase- (TDT-) mediated dUTP-biotin nick end-labeling (TUNEL) method. The expression levels of apoptosis-related proteins, such as B-cell lymphoma 2 (Bcl-2), Bcl-2 Associated X Protein (Bax), cysteine-aspartic acid protease-3 and protease-9 (caspase-3 and caspase-9), cytochrome c (cyt-C), brain-derived neurotrophic factor (BDNF), and cAMP-response element binding (CREB) protein, were evaluated by western blot. Treatment with AVLE (60 or 120 mg/kg/day) significantly improved depressive behavior. Increased apoptosis of hippocampus and cortical neurons were observed in CUMS rats, while 120 mg/kg/day of AVLE significantly reversed these changes and achieved the best antidepressant-like effects among the doses tested. Moreover, AVLE (120 mg/kg) significantly increased Bcl-2, BDNF, and CREB protein expression and decreased Bax, cyt-C, and caspase family protein expression. Our results indicate that AVLE has potent antidepressant activity, likely due to its ability to suppress neuronal apoptosis.

Abnormal modification of histone acetylation involved in depression-like behaviors of rats induced by chronically unpredicted stress.

Depression is a complex multifactorial mental disorder. Its etiology involves many factors such as social environments, genetics, and psychology. Recent studies have shown that epigenetic modification may be associated with depression. Histone acetylation is one of the main mechanisms of epigenetic modification and plays an important role in genetic expression. In this study, we investigated the role of histone acetylation in the depression-like behaviors of rats undergoing chronically unpredicted stress (CUS) by detecting the mRNA and protein expression of histone deacetylase 5, cAMP-response element-binding protein, and the level of histone acetylated modification of H3K14, H3K23, and H4K16 in the prefrontal cortex and hippocampus of the rats. The results showed that significantly increasing depression-like behaviors were observed with a decreasing histone acetylated modification level, especially on acytelated-H3K14, acytelated-H3K23, and acytelated-H4K16, upregulating histone deacetylase 5 expression and downregulating cAMP-response element-binding protein expression in CUS rats, compared with control rats. The results indicate that the decrease in the histone acetylation modification level may be partly involved in the mechanism of depression-like behaviors of rats induced by CUS.

Angelica tenuissima Nakai Ameliorates Cognitive Impairment and Promotes Neurogenesis in Mouse Model of Alzheimer's Disease.

To research Angelica tenuissima Nakai (ATN) for use in novel Alzheimer's disease (AD) therapeutics. The effect of a 30% ethanol extract of ATN (KH032) on AD-like cognitive impairment and neuropathological and neuroinflammatory changes induced by bilateral intracerebroventricular injections of β-amyloid (Aβ) peptide (Aβ1-42) was investigated. Male C57Bl/6 mice were randomly divided into 4 groups, 10 in each group. KH032-treated groups were administrated with a low or high dose of KH032 (50 and 200 mg/kg, respectively), intragastrically for 16 days; distilled water was applied in the sham and negative groups. Open fifield test, Y maze and Morris water maze test were used for behavior test and cognitive ability. In addition, the neuroprotective effects of KH032 in Aβ1-42-infused mice on the histopathological markers [neuronspecific nuclear protein (NeuN), Aβ1-42] of neurodegeneration were examined. The levels of glial fibrillary acidic protein (GFAP), NeuN, phosphorylation extracellular signal-regulated kinase (ERK)/ERK, brain-derived neurotrophic factor (BDNF), phosphorylation cAMP response element-binding (CREB)/CREB protein expression were measured by Western blot. KH032 treatment ameliorated cognitive impairments, reduced the overexpression of Aβ1-42, and inhibited neuronal loss and neuroinflammatory response in the Aβ1-42-infused mice. Moreover, KH032 treatment enhanced BDNF expression levels in the hippocampus. Finally, KH032 treatment increased phosphorylation of ERK1/2 and CREB, vital for ERK-CREB signaling. KH032 attenuated cognitive defificits in the Aβ1-42-infused mice by increasing BDNF expression and ERK1/2 and CREB phosphorylation and inhibiting neuronal loss and neuroinflflammatory response, suggesting that KH032 has therapeutic potential in neurodegenerative disorders such as AD.

Pathways Mediating Activity-Induced Enhancement of Recovery From Peripheral Nerve Injury.

This article outlines the novel hypothesis that exercise promotes axon regeneration after peripheral nerve injury through neuronal brain-derived neurotrophic factor (BDNF), and there are three required means of promoting BDNF expression: 1) increased signaling through androgen receptors, 2) increased cAMP-responsive element-binding protein expression, and 3) increased expression of the transcription factor SRY-box containing gene 11.

Signal transduction molecule patterns indicating potential glioblastoma therapy approaches

PurposeThe expression of an array of signaling molecules, along with the assessment of real-time cell proliferation, has been performed in U87 glioma cell line and in patients’ glioblastoma established cell cultures in order to provide a better understanding of cellular and molecular events involved in glioblastoma pathogenesis. Experimental therapy was performed using a phosphatidylinositol-3′-kinase (PI3K) inhibitor.Patients and methodsxMAP technology was employed to assess expression levels of several signal transduction molecules and real-time xCELLigence platform for cell behavior.ResultsPI3K inhibition induced the most significant effects on global signaling pathways in patient-derived cell cultures, especially on members of the mitogen-activated protein-kinase family, P70S6 serine-threonine kinase, and cAMP response element-binding protein expression and further prevented tumor cell proliferation.ConclusionThe PI3K pathway might be a prime target for glioblastoma treatment.

Baicalin improves chronic corticosterone-induced learning and memory deficits via the enhancement of impaired hippocampal brain-derived neurotrophic factor and cAMP response element-binding protein expression in the rat

The purpose of this study was to examine whether baicalin (BAI) improves spatial cognitive impairments induced in rats following the repeated administration of the exogenous stress hormone corticosterone (CORT). The effect of BAI on the hippocampal expression of brain-derived neurotrophic factor (BDNF) and cAMP response element-binding protein (CREB) was also investigated. For 21 days, male rats received daily doses of BAI (20, 50, and 100 mg/kg, i.p.) 1 h prior to a CORT (40 mg/kg) injection. The daily administration of BAI improved memory impairment as measured by the passive avoidance test and reduced the escape latency for finding the platform in the Morris water maze test. Additionally, as assessed by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) analysis, the administration of BAI also significantly alleviated memory-associated decreases in the expression levels of BDNF and CREB proteins and mRNAs in the hippocampus. These results demonstrate that the administration of BAI prior to high-dose exogenous CORT results in significant neuroprotective activity against neuronal impairment and memory dysfunction in rats. Thus, these findings suggest that BAI might be useful as a therapeutic agent in various neurodegenerative diseases for the improvement of cognitive function. This likely occurs through the regulation of BDNF and CREB expression.

Chronic cerebrovascular hypoperfusion affects global DNA methylation and histone acetylation in rat brain

DNA methylation and histone acetylation can be modified by various pathological or physiological factors such as hypoxia, thus influencing gene expression. In this study, we investigated the changes of global DNA methylation and histone acetylation and the related enzymes in rat brain after chronic cerebrovascular hypoperfusion by bilateral common carotid occlusion (2-VO) surgery. Colorimetric and immunohistochemistry staining were used to evaluate the global DNA methylation and histone acetylation levels, respectively. The expressions of DNA methyltransferase 1/3a (DNMT1/3a), methyl-CpG binding domain protein 2 (MBD2), histone deacetylase 3 (HDAC3) and acetyltransferase (HAT) were assessed by Western blot. We found that the level of global DNA methylation was decreased to 31.7% (P <0.01) of the sham-operated group at 10 days and increased by 30% (P <0.01) compared with the sham group at 90 days after 2-VO surgery. DNMT3a expression was down-regulated to 75.7% of the sham group, while MBD2 expression was up-regulated by 95% compared with sham group at 90 days after 2-VO. The histone H3 acetylation level was markedly decreased to 75.3% of the sham group at 10 days and 73.5% at 90 days after 2-VO, while no significant change was found for histone H4 acetylation. HDAC3 expression was markedly down-regulated to 36% of the sham group, whereas cAMP-response element binding protein expression was up-regulated by 33.6% compared with the sham group at 90 days after 2-VO. These results suggest that chronic cerebrovascular hypoperfusion influences global DNA methylation and histone acetylation levels through the related enzymes, and therefore might contribute to several neurodegenerative diseases.

Anti‐depressant Effects of Aqueous Extract from Acanthopanax senticosus in Mice

In this paper, the anti-depressant effects of Acanthopanax senticosus extract (ASE) were studied using animal models of depression including the forced swimming and tail suspension tests. The anti-depressive mechanism of ASE was explored by monitoring the levels of monoamine neurotransmitters including 5-hydroxytrylamine (5-HT), norepinephrine (NE), and dopamine (DA), as well as cAMP response element-binding (CREB) protein expression in the whole brain of mice following the tail suspension test. Our results showed that intragastric administration of ASE at a dose of 2000 mg/kg for seven days significantly reduced the duration of immobility in both the forced swimming test and the tail suspension test. These results indicate that ASE possesses antidepressant-like properties. Pre-treatment with 2000 mg/kg of ASE for seven days significantly elevated the levels of 5-HT, NE, and DA in the whole brain of mice. Moreover, ASE at doses of 1000 and 2000 mg/kg significantly up-regulated the level of CREB protein. Taken together, these findings suggest that the anti-depressive mechanism of ASE may be mediated via the central monoaminergic neurotransmitter system and CREB protein expression. Therefore, administration of ASE may be beneficial for patients with depressive disorders.

Wound-healing effect of ginsenoside Rd from leaves of Panax ginseng via cyclic AMP-dependent protein kinase pathway

Panax ginseng is considered as one of the most valuable medicinal herbs in traditional medicine, and ginsenoside Rd is one of the main active ingredients in P. ginseng leaf. Although there is significant number of evidences implicated on the beneficial effects of the ginsenosides with diverse associated mechanisms, reports on the skin regeneration by the ginsenoside Rd are not sufficient. Therefore, we examined the mitogenic and protective effects of the ginsenoside Rd in the keratinocyte progenitor cells (KPCs) and human dermal fibroblasts (HDFs). Furthermore, the signaling pathways involved in the activation of KPCs and HDFs were investigated, and wound-healing effect is evaluated in vivo through animal wound models. We found that the ginsenoside Rd significantly increased the proliferation and migration level of KPCs and HDFs in a dose-dependent manner. Additionally, the cell survival was significantly increased in H2O2 treated KPCs. Moreover, the ginsenoside Rd effectively induced collagen type 1 and down-regulated matrix metalloprotinase-1 (MMP-1) in a dose-dependent manner. All of these beneficial effects are associated with an induction of intracellular cAMP levels and phosphorylated cAMP response element-binding protein expression in nucleus, which both attenuated by adenine 9-β-d-arabinofuranoside, an adenylate cyclase inhibitor. Application of the ginsenoside Rd to an excision wound in mice showed an effective healing process. As skin regeneration is mainly associated with the activation of HDFs and KPCs, P. ginseng leaf, an alternative source of the ginsenoside Rd, can be used as a natural source for skin regeneration.

Fucoidan ameliorates scopolamine-induced neuronal impairment and memory dysfunction in rats via activation of cholinergic system and regulation of cAMP-response element-binding protein and brain-derived neurotrophic factor expressions

Effect of fucoidan (FCN) treatment on improving memory defects caused by administration of scopolamine (SCO) to the rats was examined. The effects of FCN on the acetylcholinergic system as well as the expression of cAMP-response elementbinding protein (CREB) and brain-derived neurotrophic factor (BDNF) mRNAs in the hippocampus were also investigated. Male rats were administered daily doses for 14 days of FCN (10, 20, and 50 mg/kg, i.p.) 30 min before scopolamine injection (2 mg/kg, i.p.). Daily administration of FCN improved memory impairment as measured by the passive avoidance test (PAT) and reduced the escape latency for finding the platform in the Morris water maze (MWM) test. Administration of FCN significantly alleviated memory-associated decreases in cholinergic immunoreactivity and restored the expression level of BDNF and CREB mRNAs in the hippocampus. Additionally, FCN significantly decreased the expression of pro-inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) mRNAs in the hippocampus. These results demonstrated that FCN has significant neuroprotective effects against neuronal impairment and memory dysfunction caused by scopolamine in rats. Thus, these findings suggest that FCN is useful as a therapeutic agent for improving cognitive functioning via stimulation of cholinergic enzyme activities and regulation of CREB and BDNF expressions in the brain.

Cyclic GMP/protein kinase G type‐Iα (PKG‐Iα) signaling pathway promotes CREB phosphorylation and maintains higher c‐IAP1, livin, survivin, and Mcl‐1 expression and the inhibition of PKG‐Iα kinase activity synergizes with cisplatin in non‐small cell lung cancer cells

Previously, our laboratory showed that nitric oxide (NO)/cyclic GMP (cGMP)/protein kinase G type-Iα (PKG-Iα) signaling pathway plays an important role in preventing spontaneous apoptosis and promoting cell proliferation in both normal cells (bone marrow stromal cells and vascular smooth muscle cells) and certain cancer cells (ovarian cancer cells). In the present study, we investigated the novel role of the cGMP/PKG-Iα pathway in preventing spontaneous apoptosis, promoting colony formation and regulating phosphorylation of cAMP response element binding (CREB) protein and protein expression of inhibitor of apoptosis proteins (IAPs) and anti-apoptotic Bcl-2-related proteins in NCI-H460 and A549 non-small cell lung cancer (NSCLC) cells. 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), which blocks endogenous NO-induced activation of cGMP/PKG-Iα, induced apoptosis and decreased colony formation. ODQ also decreased CREB ser133 phosphorylation and protein expression of c-IAP1, livin, and survivin. DT-2 (inhibitor of PKG-Iα kinase activity) increased apoptosis by twofold and decreased CREB ser133 phosphorylation and c-IAP1, livin, and survivin expression. Gene knockdown of PKG-Iα expression using small-interfering RNA increased apoptosis and decreased CREB ser133 phosphorylation, and c-IAP1, livin, survivin, and Mcl-1 expression. Inhibition of PKG-Iα kinase activity with DT-2 dramatically enhanced pro-apoptotic effects of the chemotherapeutic agent cisplatin. Combined treatment of DT-2 and cisplatin increased apoptosis compared with cisplatin or DT-2 alone, showing a synergistic effect. The data suggest that the PKG-Iα kinase activity is necessary for maintaining higher levels of CREB phosphorylation at ser133 and protein expression of c-IAP1, livin, survivin, and Mcl-1, preventing spontaneous apoptosis and promoting colony formation in NSCLC cells, which may limit the effectiveness of chemotherapeutic agents like cisplatin.

Targeted Deletion of LPA5 Identifies Novel Roles for Lysophosphatidic Acid Signaling in Development of Neuropathic Pain

Lysophosphatidic acid (LPA) is a bioactive lipid that serves as an extracellular signaling molecule acting through cognate G protein-coupled receptors designated LPA(1-6) that mediate a wide range of both normal and pathological effects. Previously, LPA(1), a G(αi)-coupled receptor (which also couples to other G(α) proteins) to reduce cAMP, was shown to be essential for the initiation of neuropathic pain in the partial sciatic nerve ligation (PSNL) mouse model. Subsequent gene expression studies identified LPA(5), a G(α12/13)- and G(q)-coupled receptor that increases cAMP, in a subset of dorsal root ganglion neurons and also within neurons of the spinal cord dorsal horn in a pattern complementing, yet distinct from LPA(1), suggesting its possible involvement in neuropathic pain. We therefore generated an Lpar5 null mutant by targeted deletion followed by PSNL challenge. Homozygous null mutants did not show obvious base-line phenotypic defects. However, following PSNL, LPA(5)-deficient mice were protected from developing neuropathic pain. They also showed reduced phosphorylated cAMP response element-binding protein expression within neurons of the dorsal horn despite continued up-regulation of the characteristic pain-related markers Caα(2)δ(1) and glial fibrillary acidic protein, results that were distinct from those previously observed for LPA(1) deletion. These data expand the influences of LPA signaling in neuropathic pain through a second LPA receptor subtype, LPA(5), involving a mechanistically distinct downstream signaling pathway compared with LPA(1).

Axotomy or compression is required for axonal sprouting following end-to-side neurorrhaphy

End-to-side (ETS) nerve repair remains an area of intense scrutiny for peripheral nerve surgeon-scientists. In this technique, the transected end of an injured nerve, representing the “recipient” is sutured to the side of an uninjured “donor” nerve. Some works suggest that the recipient limb is repopulated with regenerating collateral axonal sprouts from the donor nerve that go on to form functional synapses. Significant, unresolved questions include whether the donor nerve needs to be injured to facilitate regeneration, and whether a single donor neuron is capable of projecting additional axons capable of differentially innervating disparate targets. We serially imaged living transgenic mice ( n = 66) expressing spectral variants of GFP in various neuronal subsets after undergoing previously described atraumatic, compressive, or epineurotomy forms of ETS repair ( n = 22 per group). To evaluate the source, and target innervation of these regenerating axons, nerve morphometry and retrograde labeling were further supplemented by confocal microscopy as well as Western blot analysis. Either compression or epineurotomy with inevitable axotomy were required to facilitate axonal regeneration into the recipient limb. Progressively more injurious models were associated with improved recipient nerve reinnervation (epineurotomy: 184 ± 57.6 myelinated axons; compression: 78.9 ± 13.8; atraumatic: 0), increased Schwann cell proliferation (epineurotomy: 72.2% increase; compression: 39% increase) and cAMP response-element binding protein expression at the expense of a net deficit in donor axon counts distal to the repair. These differences were manifest by 150 days, at which point quantitative evidence for pruning was obtained. We conclude that ETS repair relies upon injury to the donor nerve.

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.