Cyclic Adenosine Monophosphate Analog Research Articles

Role of the cAMP-PKA-NF-κB pathway in Mucin1 over-expression in A549 cells during Respiratory syncytial virus infection

Respiratory syncytial virus (RSV) is the most common pathogen associated with acute lower respiratory tract infections in infants and young children worldwide. RSV commonly presents as bronchiolitis in young children; however, it can sometimes progress to pneumonia, respiratory failure, apnoea and even death. Although mucin1 (MUC1), a type of transmembrane glycoprotein present on airway epithelial surfaces, plays a crucial anti-inflammatory role in airway infections; however, its roles in RSV-associated acute lower respiratory tract infections have rarely been explored. In this study, we first revealed very high MUC1 protein levels in the exacerbation phase in sputum samples from children with RSV bronchiolitis. Because MUC1 is the downstream target of tumour necrosis factor-alpha (TNF-α) in RSV-infected A549 cells, we observed the inhibition of NF-κB activity, main downstream signalling of TNF-α and remarkably reduced levels of MUC1 in RSV-infected and TNF-α treated A549 cells. Furthermore, the cyclic adenosine monophosphate (cAMP) analogue (dbcAMP) downregulated the protein levels of p-IκBα and MUC1 in TNF-α-treated A549 cells. By contrast, a protein kinase A inhibitor (KT5720) up-regulated the levels of those proteins. dbcAMP and KT5720 had the same effects on MUC1 protein levels in RSV-infected A549 cells. In conclusion, we found that the cAMP-PKA-NF-κB pathway may play a role in the regulation of MUC-1 over-expression during RSV infection.

ATOH8 Expression Is Regulated by BMP2 and Plays a Key Role in Human Endometrial Stromal Cell Decidualization.

During the secretory phase of the menstrual cycle, elongated fibroblast-like mesenchymal cells in the uterine endometrium begin to transdifferentiate into polygonal epithelioid-like (decidual) cells. This decidualization process continues more broadly during early pregnancy, and the resulting decidual tissue supports successful embryo implantation and placental development. This study was carried out to determine if atonal basic helix-loop-helix transcription factor 8 (ATOH8) plays a role in human endometrial stromal fibroblast (ESF) decidualization. ATOH8 messenger RNA and protein expression levels significantly increased in human ESF cells undergoing in vitro decidualization, with the protein primarily localized to the nucleus. When ATOH8 expression was silenced, the ability of the cells to undergo decidualization was significantly diminished. Overexpression of ATOH8 enhanced the expression of many decidualization markers. Silencing the expression of ATOH8 reduced the expression of FZD4, FOXO1, and several known FOXO1-downstream targets during human ESF cell decidualization. Therefore, ATOH8 may be a major upstream regulator of the WNT/FZD-FOXO1 pathway, previously shown to be critical for human endometrial decidualization. Finally, we explored possible regulators of ATOH8 expression during human ESF decidualization. BMP2 significantly enhanced ATOH8 expression when cells were stimulated to undergo decidualization, while an ALK2/3 inhibitor reduced ATOH8 expression. Finally, although the steroids progesterone plus estradiol did not affect ATOH8 expression, the addition of cyclic adenosine monophosphate (cAMP) analogue alone represented the major effect of ATOH8 expression when cells were stimulated to undergo decidualization. Our results suggest that ATOH8 plays a crucial role in human ESF decidualization and that BMP2 plus cAMP are major regulators of ATOH8 expression.

Luteal Lipid Droplets: A Novel Platform for Steroid Synthesis.

Progesterone is an essential steroid hormone that is required to initiate and maintain pregnancy in mammals and serves as a metabolic intermediate in the synthesis of endogenously produced steroids, including sex hormones and corticosteroids. Steroidogenic luteal cells of the corpus luteum have the tremendous capacity to synthesize progesterone. These specialized cells are highly enriched with lipid droplets that store lipid substrate, which can be used for the synthesis of steroids. We recently reported that hormone-stimulated progesterone synthesis by luteal cells requires protein kinase A-dependent mobilization of cholesterol substrate from lipid droplets to mitochondria. We hypothesize that luteal lipid droplets are enriched with steroidogenic enzymes and facilitate the synthesis of steroids in the corpus luteum. In the present study, we analyzed the lipid droplet proteome, conducted the first proteomic analysis of lipid droplets under acute cyclic adenosine monophosphate (cAMP)-stimulated conditions, and determined how specific lipid droplet proteins affect steroidogenesis. Steroidogenic enzymes, cytochrome P450 family 11 subfamily A member 1 and 3 beta-hydroxysteroid dehydrogenase (HSD3B), were highly abundant on lipid droplets of the bovine corpus luteum. High-resolution confocal microscopy confirmed the presence of active HSD3B on the surface of luteal lipid droplets. We report that luteal lipid droplets have the capacity to synthesize progesterone from pregnenolone. Lastly, we analyzed the lipid droplet proteome following acute stimulation with cAMP analog, 8-Br-cAMP, and report increased association of HSD3B with luteal lipid droplets following stimulation. These findings provide novel insights into the role of luteal lipid droplets in steroid synthesis.

Cyclic adenosine monophosphate (cAMP) analog and phosphodiesterase inhibitor (IBMX) ameliorate human sperm capacitation and motility

ObjectivesHuman sperm quality is decreasing progressively. One of the foremost reasons for infertility is the failure in sperm capacitation. We examined the influence of a cAMP (cyclic-adenosine mono phosphate analog)+IBMX (3-isobutyl-1-methylxanthine) on the motility and capacitation rate of human sperm over time. Material and methodsSamples were gotten from 20 asthenozoospermic infertile patients referring to the Academic Center for Education, Culture and Research unit of the infertility research center, Qom, Iran. Samples were processed with a Density Gradient Centrifuging. Spermatozoa were divided into 4 groups: control, experimental 1, 2 and 3 (E1, E2, E3) based on the dose/time schedules (cAMP 5mmol+IBMX 0.2mmol/2, 4, and 6h, respectively). The computer-assisted sperm analysis and chlortetracycline assays were used to measure sperm motility and capacitation. ResultsAfter incubation with a cAMP analog and IBMX, the levels of progressive motile sperms considerably improved in all experimental groups compared to the control group (E1=18.89±7.1, E2=30±9.7, E3=26.3±9.6 vs Control=10.28±6.2, P<0.05) especially in E2 group (P<0.05), indicating a greater effect of db cAMP (5mmol) and IBMX (0.2mmol) for 4h compared to the same doses at 2 and 6h. Also, non-progressive motile sperms significantly decreased in E2 group compared to the other groups (P<0.05). Moreover, both patterns C and B were substantially improved in all experimental groups especially in E2 group (P<0.05). ConclusionOur findings support that the supplementation of sperm with db cAMP+IBMX specially for 4h, could be useful for men with asthenozoospermia to improve the success of assisted reproductive technology.

The role of mitochondria in the recovery of neurons after injury.

The role of mitochondria in the recovery of neurons after injury.

Supplementation of Freezing Media with Cyclic Adenosine Monophosphate Analog and Isobutylmethylxanthine on Sperm Quality

Background: This study aimed to explore whether the addition of a cyclic adenosine monophosphate (cAMP) analog and isobutylmethylxanthine (IBMX) in freezing media improved sperm quality and what role cAMP has in this recovery. Materials and methods: ach semen sample was cryopreserved into four groups: fresh semen sample, as a control group, freezing medium + 2.5 mM cAMP analog and 0.2 mM IBMX, freezing medium + 12.5 mM cAMP analog and 0.2 mM IBMX, and freezing medium + 25 mM cAMP analog and 0.2 mM IBMX. Sperm parameters after post-thaw were analyzed according to WHO instruction (2010). Viability, acrosome reaction, and DNA damage levels of the samples were evaluated. Results: Our results indicated that the effective concentrations of 12.5 and 25 mM cAMP analog and 0.2 mM IBMX significantly improved the total motility, progressive motility, and viability of the frozen-thawed (P&lt;0.05). However, non-progressive motility and immotile were significantly reduced in the 12.5 and 25 mM cAMP analogs and 0.2 mM IBMX groups after thawing (P&lt;0.05). During freezing the spermatozoa, the high concentration of the cAMP analog increased acrosome reaction after thawing in the 25 mM and 0.2 mM IBMX treated samples (P&lt;0.05). DNA fragmentation in 25 mM cAMP analog and 0.2 mM (IBMX) supplementation was significantly lower compared to the other groups (P&lt;0.05). Conclusions: Our findings revealed that in vitro cAMP analog and IBMX supplementation in freezing media play an important role in preventing cryodamage by maintaining the sperm functional parameters.

Human brain microvascular endothelial cell pairs model tissue-level blood-brain barrier function.

The blood-brain barrier plays a critical role in delivering oxygen and nutrients to the brain while preventing the transport of neurotoxins. Predicting the ability of potential therapeutics and neurotoxicants to modulate brain barrier function remains a challenge due to limited spatial resolution and geometric constraints offered by existing in vitro models. Using soft lithography to control the shape of microvascular tissues, we predicted blood-brain barrier permeability states based on structural changes in human brain endothelial cells. We quantified morphological differences in nuclear, junction, and cytoskeletal proteins that influence, or indicate, barrier permeability. We established a correlation between brain endothelial cell pair structure and permeability by treating cell pairs and tissues with known cytoskeleton-modulating agents, including a Rho activator, a Rho inhibitor, and a cyclic adenosine monophosphate analog. Using this approach, we found that high-permeability cell pairs showed nuclear elongation, loss of junction proteins, and increased actin stress fiber formation, which were indicative of increased contractility. We measured traction forces generated by high- and low-permeability pairs, finding that higher stress at the intercellular junction contributes to barrier leakiness. We further tested the applicability of this platform to predict modulations in brain endothelial permeability by exposing cell pairs to engineered nanomaterials, including gold, silver-silica, and cerium oxide nanoparticles, thereby uncovering new insights into the mechanism of nanoparticle-mediated barrier disruption. Overall, we confirm the utility of this platform to assess the multiscale impact of pharmacological agents or environmental toxicants on blood-brain barrier integrity.

DBcAMP Rescues the Neurons From Degeneration in Kainic Acid-Injured Hippocampus, Enhances Neurogenesis, Learning, and Memory.

Dibutyryl cyclic adenosine monophosphate (dBcAMP) is a cell-permeable synthetic analog of cyclic adenosine monophosphate (cAMP). Although the elevation of cAMP levels was reported to promote the functional recovery in spinal cord injury, its role in neurogenesis or functional recovery after hippocampal injury is unknown. The objective of the study was to investigate the effects of dBcAMP on learning, memory, and hippocampal neurogenesis in the excitotoxically lesioned hippocampus. An excitotoxic lesion was induced in the hippocampi of 4-month-old male BALB/c mice by injecting 0.25 μg/μl into the lateral ventricles of both sides. The lesioned mice (L) were divided into L+dBcAMP and L+phosphate-buffered saline (PBS) groups. Sham surgery (S) was done by the injection of 1 μl of sterile saline into the lateral ventricles. The sham surgery mice were divided into S+dBcAMP and S+PBS groups. Mice in the L+dBcAMP and S+dBcAMP groups were treated with dBcAMP for 1 week (i.p., 50 mg/kg), whereas mice in the L+PBS and S+PBS groups were treated with PBS. The mice in all groups were subjected to water maze and passive avoidance tests at the end of the 4th week. Cresyl violet staining and NeuN and doublecortin immunostaining were done to analyze the morphology and neurogenesis. The water maze learning sessions did not show a significant difference in escape latency between the groups, suggesting an unimpaired learning ability of mice in all groups. The L+dBcAMP mice had significantly short entry latency and higher target quadrant time/distance traveled compared to the L+PBS group, suggesting better memory retention. The L+dBcAMP group had a significantly improved memory retention compared to the L+PBS mice during the passive avoidance test. Morphological studies showed significantly greater adult neurons and increased hippocampal neurogenesis in the hippocampus of mice in the L+dBcAMP group compared to those in the L+PBS group. There was no significant difference between the S+dBcAMP and S+PBS groups in the water maze/passive avoidance tests and the number of neurons. In conclusion, dBcAMP protects the hippocampal neuron from degeneration and enhances hippocampal neurogenesis, learning, and memory.

Dibutyryl cAMP Enhances Cognitive Functions in Diabetic Rats by Increasing the Hippocampal Neurogenesis

Dibutyryl cyclic adenosine monophosphate (dBcAMP) is a cell‐permeable synthetic analog of cyclic adenosine monophosphate (cAMP) known to acts as an intracellular second messenger for neurotransmitters and hormones. dBcAMP enhances differentiation and survival of neurons from neuronal stem cells, differentiation of mesenchymal stromal cell into neurons and stimulates the differentiation of astrocytes and contribute to neuroprotection in neural tissue injury.Diabetes, a chronic metabolic disease has adverse effects on adult hippocampal neurogenesis and cognitive function. Even though, dBcAMP is used as a therapeutic agent in some disease models, it has not been used in diabetes. Present experiment was aimed to study the effects of dBcAMP on learning and memory and adult neurogenesis, in streptozotocin model of diabetic rats. Diabetes was induced in male Wistar rats (3 months old) by injecting streptozotocin (ip, 50mg/kg) into the peritoneal cavity. After confirming the diabetic condition (hyperglycemia) in these rats after 48hrs of STZ injection, they were grouped into diabetic (DI) and diabetic+dBcAMP (DI+dBcAMP groups. Age matched normal rats served as control group (C). DI+dBcAMP group were treated with dBcAMP (ip,10mg/kg) intraperitoneally for 10 days and C and DI groups were be treated with PBS for the same duration. After treatment, animals in all groups were subjected to learning and memory test in Morris water maze from day21 to day27 of the experiment to assess the cognitive function. After the behavioral test rats (n=6 in all groups) were sacrificed on day 30. Fresh hippocampal tissue were collected for ELISA (for BDNF, brain derived neurotrophic factor), and Western blot (for doublecortin, DCX) analysis. Rats (n=6 in all groups), were be perfused with 4% paraformaldehyde and hippocampi were collected for immunostaining (DCX). Number of DCX positive neurons were quantified in the hippocampal dentate gyrus. Data were analyzed by One/Two –way ANOVA, followed by Bonferroni's multiple comparison test. Results showed a significant memory deficit (decreased time spent and distance traveled in platform quadrant) in DI rats compared to C (p&lt;0.001, One way ANOVA), and memory was significantly increased in DI+ dBcAMP compared to DI group (p&lt;0.001). Analysis of number of DCX positive neurons in the dentate gyrus (neurogenesis) showed a significant decrease in neurogenesis in DI group compared to C group (p&lt;0.001, One‐way ANOVA) and it was significantly increased in DI+dBcAMP (p&lt;0.001, One‐way ANOVA). Western blot analysis for DCX showed significant increase in DI+ dBcAMP group (p&lt;0.01) and hence confirmed the DCX positive neuron count data. Analysis of BDNF levels in the hippocampus of the DI+dBcAMP was found to be significantly increased compared to DI group (p&lt;0.01). We conclude that dBcAMP enhances cognitive functions in diabetic rats by increasing the Hippocampal BDNF levels and hence enhanced neurogenesis. Thus dBcAMP may be a potential molecule to be considered in treatment of diabetic patients to prevent the cognitive deterioration.Support or Funding InformationCollege of Graduate Studies and Research Sector, Kuwait UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Oral administration of dibutyryl adenosine cyclophosphate improved growth performance in weaning piglets by enhancing lipid fatty acids metabolism

Dibutyryl adenosine cyclophosphate (dbcAMP-Ca), an analog of cyclic adenosine monophosphate (cAMP), plays greater roles in regulating physiological activities and energy metabolism than cAMP. The aim of this study was to investigate the effect of oral administration of dbcAMP-Ca on growth performance and fatty acids metabolism in weaning piglets. A total of 14 early weaning piglets (7 ± 1 d of age, 3.31 ± 0.09 kg, Landrace × Large White × Duroc) were randomly divided into 2 groups: control group and dbcAMP-Ca group, and the piglets received 7 mL of 0.9% NaCl or 1.5 mg dbcAMP-Ca dissolved in 7 mL of 0.9% NaCl per day for 10 d, respectively. The results showed that the average daily gain (ADG) increased by 109.17% (P < 0.05) in the dbcAMP-Ca group compared with the control group. Besides, dbcAMP-Ca significantly decreased blood high density lipoprotein cholesterol (HDLC) concentration (P < 0.05) and significantly increased blood low density lipoprotein cholesterol (LDLC) concentration (P ＜ 0.05) compared with the control group. Further, liver C18:2n6t content significantly increased in dbcAMP-Ca group (P < 0.05) compared with the control group. With the increase of C18:2n6t content, the mRNA expression levels of peroxisome proliferator-activated receptor α (PPARα) and hormone sensitive glycerol three lipase (HSL), of which genes are related to lipid metabolism, were also significantly increased (P < 0.05 or P < 0.01). All of the results indicated that dbcAMP-Ca improved the ADG, which was probably done by regulating fatty acids metabolism in the liver of weaning piglets.

Inhibition of protein kinase A and GIRK channel reverses fentanyl-induced respiratory depression

Opioid-induced respiratory depression is a major obstacle to improving the clinical management of moderate to severe chronic pain. Opioids inhibit neuronal activity via various pathways, including calcium channels, adenylyl cyclase, and potassium channels. Currently, the underlying molecular pathway of opioid-induced respiratory depression is only partially understood. This study aimed to investigate the mechanisms of opioid-induced respiratory depression in vivo by examining the effects of different pharmacological agents on fentanyl-induced respiratory depression. Respiratory parameters were detected using whole body plethysmography in conscious rats. We show that pre-treatment with the protein kinase A (PKA) inhibitor H89 reversed the fentanyl-related effects on respiratory rate, inspiratory time, and expiratory time. Pre-treatment with the G protein-gated inwardly rectifying potassium (GIRK) channel blocker Tertiapin-Q dose-dependently reversed the fentanyl-related effects on respiratory rate and inspiratory time. A phosphodiesterase 4 (PDE4) inhibitor and cyclic adenosine monophosphate (cAMP) analogs did not affect fentanyl-induced respiratory depression. These findings suggest that PKA and GIRK may be involved in fentanyl-induced respiratory depression and could represent useful therapeutic targets for the treatment of fentanyl-induced ventilatory depression.

Scalable Differentiation and Dedifferentiation Assays Using Neuron-Free Schwann Cell Cultures.

This chapter describes protocols to establish simplified in vitro assays of Schwann cell (SC) differentiation in the absence of neurons. The assays are based on the capacity of isolated primary SCs to increase or decrease the expression of myelination-associated genes in response to the presence or absence of cell permeable analogs of cyclic adenosine monophosphate (cAMP). No special conditions of media or substrates beyond the administration or removal of cAMP analogs are required to obtain a synchronous response on differentiation and dedifferentiation. The assays are cost-effective and far easier to implement than traditional myelinating SC-neuron cultures. They are scalable to a variety of plate formats suited for downstream experimentation and analysis. These cell-based assays can be used as drug discovery platforms for the evaluation of novel agents controlling the onset, maintenance, and reversal of the differentiated state using any typical adherent SC population.

Physiological characterization and regulation of the contractile properties of the mosquito ventral diverticulum (crop)

In adult dipteran insects (flies), the crop is a diverticulum of the esophagus that serves as a food storage organ. The crop pumps stored contents into the alimentary canal for digestion and absorption. The pumping is mediated by peristaltic contractions of the crop musculature. In adult female mosquitoes, the crop (ventral diverticulum) selectively stores sugar solutions (e.g., nectar); proteinaceous blood meals by-pass the crop and are transferred directly to the midgut for digestion. The mechanisms that regulate crop contractions have never been investigated in mosquitoes. Here we provide the first physiological characterization of the contractile properties of the mosquito crop and explore the mechanisms that regulate crop contractions. Using an in vitro bioassay we found that the isolated crop spontaneously contracts in Ringer solution for at least 1 h and its contractions are dependent on extracellular Ca2+. Adding serotonin (5-hydroxytryptamine, 5-HT) or a membrane-permeable analog of cyclic adenosine monophosphate (cAMP) to the extracellular bath increased the frequency of crop contractions. On the other hand, adding benzethonium chloride (BzCl; a chemical that mimics the effects of myosuppressins), H-89 or Rp-cAMPS (inhibitors of protein kinase A, PKA), or carbenoxolone (an inhibitor of gap junctions) reduced the frequency of the unstimulated, spontaneous and/or 5-HT-stimulated crop contractions. Adding aedeskinin III did not detectably alter crop contraction rates. In addition to pharmacological evidence of gap junctions, we demonstrated that the crop expressed several mRNAs encoding gap junctional proteins (i.e. innexins). Furthermore, we localized immunoreactivity for innexin 2 and innexin 3 to muscle and epithelial cells of the crop, respectively. Our results 1) suggest that 5-HT and myosupressins oppositely regulate contractile activity of the mosquito crop, and 2) provide the first evidence for putative roles of cAMP, PKA, and gap junctions in modulating contractile activity of the dipteran crop.

Effect of bucladesine, pentoxifylline, and H-89 as cyclic adenosine monophosphate analog, phosphodiesterase, and protein kinase A inhibitor on acute pain.

The aim of this study was to determine the effects of cyclic adenosine monophosphate (cAMP) and its dependent pathway on thermal nociception in a mouse model of acute pain. Here, we studied the effect of H-89 (protein kinase A inhibitor), bucladesine (Db-cAMP) (membrane-permeable analog of cAMP), and pentoxifylline (PTX; nonspecific phosphodiesterase (PDE) inhibitor) on pain sensation. Different doses of H-89 (0.05, 0.1, and 0.5 mg/100 g), PTX (5, 10, and 20 mg/100 g), and Db-cAMP (50, 100, and 300 nm/mouse) were administered intraperitoneally (I.p.) 15 min before a tail-flick test. In combination groups, we injected the first and the second compounds 30 and 15 min before the tail-flick test, respectively. I.p. administration of H-89 and PTX significantly decreased the thermal-induced pain sensation in their low applied doses. Db-cAMP, however, decreased the pain sensation in a dose-dependent manner. The highest applied dose of H-89 (0.5 mg/100 g) attenuated the antinociceptive effect of Db-cAMP in doses of 50 and 100 nm/mouse. Surprisingly, Db-cAMP decreased the antinociceptive effect of the lowest dose of H-89 (0.05 mg/100 g). All applied doses of PTX reduced the effect of 0.05 mg/100 g H-89 on pain sensation; however, the highest dose of H-89 compromised the antinociceptive effect of 20 mg/100 g dose of PTX. Co-administration of Db-cAMP and PTX increased the antinociceptive effect of each compound on thermal-induced pain. In conclusion, PTX, H-89, and Db-cAMP affect the thermal-induced pain by probably interacting with intracellular cAMP and cGMP signaling pathways and cyclic nucleotide-dependent protein kinases.

A Vascular Permeability Assay Using an In Vitro Human Microvessel Model Mimicking the Inflammatory Condition.

The vascular barrier is an important function of the endothelium and its dysfunction is involved in several diseases. The barrier function of the endothelial cell monolayer is governed by cell-cell, cell-extracellular matrix (cell-ECM) contacts, and inflammatory factors such as thrombin, histamine or vascular endothelial growth factor. Several in vivo and in vitro assays that measure the vascular permeability induced by these factors have been developed. However, they suffer limitations such as being challenging for assessing details of biological processes at a cellular level or lacking the architecture of a vessel, that raise the need for new methods. In vitro 3D model-based assays have thus been developed but assays for investigating compounds that protects the barrier function are lacking. Here we describe the development of an in vitro three-dimensional (3D) vascular endothelium model in which we can manipulate the endothelial barrier function and permeability to molecules, which have a molecular weight similar to human serum albumin, allowing to assess the protective effect of compounds. A microvessel was prepared by culturing human umbilical vein endothelial cells (HUVECs) within a collagen gel on a polydimethylsiloxane (PDMS) chip. Using fluorescein isothiocyanate (FITC)-conjugated dextran (70 kDa, FITC-dextran) and confocal fluorescence microscopy, we showed that the microvessel presented an effective barrier function. We were then able to induce the loss of this barrier function by treatment with the inflammatory factor thrombin. The loss of barrier function was quantified by the extravasation of FITC-dextran into collagen matrix. Furthermore, we were able to analyze the protective effect on the endothelial barrier function of the cyclic adenosine monophosphate (cAMP) analog, 8-pCPT-2'-O-Me-cAMP (also called 007). In an attempt to understand the effects of thrombin and 007 in our model, we analyzed the adherens junctions and cytoskeleton through immunostaining of the vascular endothelial cadherin and actin, respectively. Our assay method could be used to screen for compounds modulating the barrier function of endothelial cells, as well as investigating mechanistic aspects of barrier dysfunction.

Lithium Reversibly Inhibits Schwann Cell Proliferation and Differentiation Without Inducing Myelin Loss.

This study was undertaken to examine the bioactivity, specificity, and reversibility of lithium's action on the growth, survival, proliferation, and differentiation of cultured Schwann cells (SCs). In isolated SCs, lithium promoted a state of cell cycle arrest that featured extensive cell enlargement and c-Jun downregulation in the absence of increased expression of myelin-associated markers. In addition, lithium effectively prevented mitogen-induced S-phase entry without impairing cell viability. When lithium was administered together with differentiating concentrations of cyclic adenosine monophosphate (cAMP) analogs, a dramatic inhibition of the expression of the master regulator of myelination Krox-20 was observed. Likewise, lithium antagonized the cAMP-dependent expression of various myelin markers such as protein zero, periaxin, and galactocerebroside and allowed SCs to maintain high levels of expression of immature SC markers even in the presence of high levels of cAMP and low levels of c-Jun. Most importantly, the inhibitory action of lithium on SC proliferation and differentiation was shown to be dose dependent, specific, and reversible upon removal of lithium compounds. In SC-neuron cultures, lithium suppressed myelin sheath formation while preserving axonal integrity, SC-axon contact, and basal lamina formation. Lithium was unique in its ability to prevent the onset of myelination without promoting myelin degradation or SC dedifferentiation. To conclude, our results underscored an unexpected antagonistic action of lithium on SC mitogenesis and myelin gene expression. We suggest that lithium represents an attractive pharmacological agent to safely and reversibly suppress the onset of SC proliferation, differentiation, and myelination while maintaining the integrity of pre-existing myelinated fibers.

Triple negative breast cancer development can be selectively suppressed by sustaining an elevated level of cellular cyclic AMP through simultaneously blocking its efflux and decomposition.

Triple negative breast cancer (TNBC) has the highest mortality among all breast cancer types and lack of targeted therapy is a key factor contributing to its high mortality rate. In this study, we show that 8-bromo-cAMP, a cyclic adenosine monophosphate (cAMP) analog at high concentration (> 1 mM) selectively suppresses TNBC cell growth. However, commonly-used cAMP-elevating agents such as adenylyl cyclase activator forskolin and pan phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) are ineffective. Inability of cAMP elevating agents to inhibit TNBC cell growth is due to rapid diminution of cellular cAMP through efflux and decomposition. By performing bioinformatics analyses with publically available gene expression datasets from breast cancer patients/established breast cancer cell lines and further validating using specific inhibitors/siRNAs, we reveal that multidrug resistance-associated protein 1/4 (MRP1/4) mediate rapid cAMP efflux while members PDE4 subfamily facilitate cAMP decomposition. When cAMP clearance is prevented by specific inhibitors, forskolin blocks TNBC's in vitro cell growth by arresting cell cycle at G1/S phase. Importantly, cocktail of forskolin, MRP inhibitor probenecid and PDE4 inhibitor rolipram suppresses TNBC in vivo tumor development. This study suggests that a TNBC-targeted therapeutic strategy can be developed by sustaining an elevated level of cAMP through simultaneously blocking its efflux and decomposition.

8-Cl-cAMP and PKA I-selective cAMP analogs effectively inhibit undifferentiated thyroid cancer cell growth.

The main purpose of our work was to evaluate the effects of different cyclic adenosine monophosphate analogs on thyroid cancer-derived cell lines. In particular we studied 8-chloroadenosine-3',5'-cyclic monophosphate, the most powerful cyclic adenosine monophosphate analog, and the protein kinase A I-selective combination of 8-hexylaminoadenosine-3',5'cyclic monophosphate and 8-piperidinoadenosine-3',5'-cyclic monophosphate. The cyclic adenosine monophosphate/protein kinase A pathway plays a fundamental role in the regulation of thyroid cells growth. Site-selective cyclic adenosine monophosphate analogs are a class of cyclic adenosine monophosphate-derivate molecules that has been synthesized to modulate protein kinase A activity. Although the cyclic adenosine monophosphate/protein kinase A pathway plays a fundamental role in the regulation of thyroid cells proliferation, there are currently no studies exploring the role of cyclic adenosine monophosphate analogs in thyroid cancer. We evaluated the effects on cell proliferation, apoptosis activation and alterations of different intracellular pathways using 3-(4,5-dimetylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytofluorimetry, western blotting, and kinase inhibitors. Our results show that both compounds have antiproliferative potential. Both treatments were able to modify protein kinase A RI/RII ratio, thus negatively influencing cancer cells growth. Moreover, the two treatments differentially modulated various signaling pathways that regulate cell proliferation and apoptosis. Both treatments demonstrated interesting characteristics that prompt further studies aiming to understand the intimate interaction between different intracellular pathways and possibly develop novel anticancer therapies for undifferentiated thyroid cancer.

Protein Kinase A Signaling Is Inhibitory for Reprogramming into Pluripotent Stem Cells.

Somatic cells may be reprogrammed into pluripotent cells by the ectopic expression of defined transcription factors. However, some of the hurdles that affect the generation of induced pluripotent stem cells include extremely low efficiency and slow reprogramming. In the present study, we examined the effects of small molecules on cellular reprogramming and found that 8-Bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), an analog of cyclic adenosine monophosphate (cAMP), improves the reprogramming efficiency of reprogrammable mouse fibroblasts induced with dox in serum replacement (SR) medium. Interestingly, treatment with 8-Br-cAMP in mouse embryonic stem cell culture conditions does not affect reprogramming into the pluripotent state; however, reprogramming efficiency is significantly enhanced by inhibition of protein kinase A (PKA) in SR medium. Therefore, our results suggest that PKA signaling is unnecessary and may in fact act as a barrier to reprogramming into pluripotent stem cells.

MicroRNA-132 promotes estradiol synthesis in ovarian granulosa cells via translational repression of Nurr1.

BackgroundEstrogen synthesis is an important function of the mammalian ovary. Estrogen plays important roles in many biological processes, including follicular development, oocyte maturation and endometrial proliferation, and dysfunctions in estrogen synthesis contribute to the development of polycystic ovary syndrome and premature ovarian failure. Classical signaling cascades triggered by follicle-stimulating hormone induce estrogen synthesis via the upregulation of Cyp19a1 in granulosa cells (GCs). This study aimed to determine the effect of microRNA-132 (miR-132) on estradiol synthesis in GCs.MethodsPrimary mouse GCs were collected from ovaries of 21-day-old immature ICR mice through follicle puncture. GCs were cultured and treated with the stable cyclic adenosine monophosphate analog 8-Br-cAMP or transfected with miR-132 mimics, Nurr1-specific small interfering RNA oligonucleotides and Flag-Nurr1 plasmids. Concentrations of estradiol and progesterone in culture medium were determined by an automated chemiluminescence-based assay. Quantitative real time PCR and western blot were performed to identify the effect of miR-132 on Cyp19a1, Cyp11a1 and an orphan nuclear receptor-Nurr1 expression in GCs. Direct suppression of Nurr1 via its 3'-untranslated region by miR-132 were further verified using luciferase reporter assays.ResultsThe expression level of miR-132 in cultured mouse GCs was significantly elevated during 48 h of treatment with 8-Br-cAMP. The synthesis of estradiol increased after the overexpression of miR-132 in mouse GCs. The real-time PCR results demonstrated that miR-132 induced the expression of Cyp19a1 significantly. Nurr1, an orphan nuclear receptor that suppresses Cyp19a1 expression, was found to be a direct target of miR-132. Nurr1 was suppressed by miR-132, as indicated by a luciferase assay and Western blotting. The knockdown of Nurr1 primarily elevated the synthesis of estradiol and partially attenuated the miR-132-induced estradiol elevation, and the ectopic expression of Flag-Nurr1 abrogated the stimulatory effect of miR-132 on estradiol synthesis in mouse GCs.ConclusionsOur findings suggest that miR-132 is involved in the cAMP signaling pathway and promotes estradiol synthesis via the translational repression of Nurr1 in ovarian GCs.