cAMP Pathway Research Articles

Nardosinone regulates the slc38a2 gene to alleviate Parkinson's symptoms in rats through the GABAergic synaptic and cAMP pathways

In a rotenone-induced Parkinson’s disease (PD) rat model, behavioral investigation, pathological examination, inflammatory factor analysis, and mitochondrial structure and function investigation verified the anti-PD efficacy of nardosinone. A combined transcriptome and proteome analysis proposed that the anti-PD target of nardosinone is the slc38a2 gene and may involve the GABAergic synaptic pathway and cAMP-signaling pathway. Analysis of targeted slc38a2 knockout cells and expression of key enzyme-encoding genes in both pathways verified the target and pathways proposed by the ‘omics analysis. This further confirms that nardosinone can regulate the slc38a2 gene, a potential new target for the treatment of Parkinson's disease, and plays an anti-PD role through the GABAergic synaptic and cAMP pathways.

Raf Kinase Inhibitory Protein regulates the cAMP-dependent protein kinase signaling pathway through a positive feedback loop

Raf Kinase Inhibitory Protein (RKIP) maintains cellular robustness and prevents the progression of diseases such as cancer and heart disease by regulating key kinase cascades including MAP kinase and protein kinase A (PKA). Phosphorylation of RKIP at S153 by Protein Kinase C (PKC) triggers a switch from inhibition of Raf to inhibition of the G protein coupled receptor kinase 2 (GRK2), enhancing signaling by the β-adrenergic receptor (β-AR) that activates PKA. Here we report that PKA-phosphorylated RKIP promotes β-AR-activated PKA signaling. Using biochemical, genetic, and biophysical approaches, we show that PKA phosphorylates RKIP at S51, increasing S153 phosphorylation by PKC and thereby triggering feedback activation of PKA. The S51V mutation blocks the ability of RKIP to activate PKA in prostate cancer cells and to induce contraction in primary cardiac myocytes in response to the β-AR activator isoproterenol, illustrating the functional importance of this positive feedback circuit. As previously shown for other kinases, phosphorylation of RKIP at S51 by PKA is enhanced upon RKIP destabilization by the P74L mutation. These results suggest that PKA phosphorylation at S51 may lead to allosteric changes associated with a higher-energy RKIP state that potentiates phosphorylation of RKIP at other key sites. This allosteric regulatory mechanism may have therapeutic potential for regulating PKA signaling in disease states.

The genomic and transcriptome characteristics of lung adenocarcinoma patients with previous breast cancer

BackgroundBreast cancer and lung cancer are the top two malignancies in the female population and the number of patients with breast cancer and subsequent primary lung cancer has increased significantly in recent years. However, the unique molecular characteristics of this group of patients remains unclear.PurposeTo identify the genomic and transcriptome characteristics of primary lung adenocarcinoma patients with previous breast cancer by comparison with single primary lung adenocarcinoma (SPLA) patients.MethodsThe tumor and normal pulmonary tissue specimens of ten primary pulmonary adenocarcinoma patients with previous breast cancer (multiple primary cancer, MPC) and ten SPLA patients were prospectively collected. The whole exome sequencing (WES) and RNA sequencing (RNA-seq) were performed to analyze the gene mutation and expression differences between MPC and SPC patients.ResultsThe results of WES indicated that the mutations of TRIM73, DLX6 and CNGB1 only existed in MPC patients. The results of RNA-seq manifested the occurrence of second primary lung adenocarcinoma in breast cancer patients was closely associated with cytokine-cytokine receptor action, autophagy, PI3L-Akt, cAMP and calcium ion signaling pathways. Besides, the expression levels of FGF10 and VEGFA genes were significantly increased in MPC patients.ConclusionThe occurrence of second primary lung adenocarcinoma may be related to the cytokine-cytokine receptor action, autophagy, PI3L-Akt, cAMP and calcium ion signaling pathways. Furthermore, the mutations of TRIM73, DLX6 and CNGB1 and high expression of FGF10 and VEGFA might play an important role in the development of lung adenocarcinoma in breast cancer patients. However, more in-depth investigations are needed to verify above findings.

Purkinje cell-specific deletion of CREB worsens alcohol-induced cerebellar neuronal losses and motor deficits.

Exposure to alcohol during pregnancy can kill developing fetal neurons and lead to fetal alcohol spectrum disorder (FASD) in the offspring. However, not all fetuses are equally vulnerable to alcohol toxicity. These differences in vulnerability among individuals are likely due, at least in part, to genetic differences. Some genes encode neuroprotective molecules that act through signaling pathways to protect neurons against alcohol's toxic effects. One signaling pathway that can protect cultured neurons against alcohol-induced cell death invitro is the cAMP pathway. A goal of this study was to determine whether the cAMP pathway can exert a similar neuroprotective effect against alcohol invivo. A key molecule within the cAMP pathway is cAMP response element binding protein (CREB). In this study, CREB was specifically disrupted in cerebellar Purkinje cells to study its role in protection of cerebellar neurons against alcohol toxicity. Mice with Purkinje cell-specific knockout of CREB were generated with the Cre-lox system. A 2×2 design was used in which Cre-negative and Cre-positive mice received either 0.0 or 2.2mg/g ethanol by intraperitoneal (i.p.) injection daily over postnatal day (PD) 4-9. Stereological cell counts of cerebellar Purkinje cells and granule cells were performed on PD 10. Motor function was assessed on PD 40 using the rotarod. Purkinje cell-specific disruption of CREB alone (in the absence of alcohol) induced only a small reduction in Purkinje cell number. However, the loss of CREB function from Purkinje cells greatly increased the vulnerability of Purkinje cells to alcohol-induced cell death. While alcohol killed 20% of Purkinje cells in the Cre-negative (CREB-expressing) mice, alcohol killed 57% of Purkinje cells in the Cre-positive (CREB-nonexpressing) mice. This large loss of Purkinje cells did not lead to similar alcohol-induced losses of granule cells. In the absence of alcohol, lack of CREB function in Purkinje cells had no effect on rotarod performance. However, in the presence of alcohol, disruption of CREB in Purkinje cells substantially worsened rotarod performance. Disruption of a single gene (CREB) in a single neuronal population (Purkinje cells) greatly increases the vulnerability of that cell population to alcohol-induced cell death and worsens alcohol-induced brain dysfunction. The results suggest that the cAMP pathway can protect cells invivo against alcohol toxicity and underline the importance of genetics in determining the neuropathology and behavioral deficits of FASD.

Cyclic Adenosine Monophosphate (cAMP)-Dependent Phosphodiesterase Inhibition Promotes Bone Anabolism Through CD8+ T Cell Wnt-10b Production in Mice.

ABSTRACTCyclic adenosine monophosphate (cAMP)‐dependent phosphodiesterase (PDE) inhibitors such as pentoxifylline (PTX) suppress cAMP degradation and promote cAMP‐dependent signal transduction. PDE inhibitors increase bone formation and bone mass in preclinical models and are used clinically to treat psoriatic arthritis by targeting inflammatory mediators including activated T cells. T cell activation requires two signals: antigen‐dependent CD3‐activation, which stimulates cAMP production; and CD28 co‐stimulation, which downregulates cAMP‐signaling, through PDE activation. PDE‐inhibitors consequently suppress T cell activation by disrupting CD28 co‐stimulation. Interestingly, we have reported that when CD8+ T cells are activated in the absence of CD28 co‐stimulation, they secrete Wnt‐10b, a bone anabolic Wnt ligand that promotes bone formation. In the present study, we investigated whether the bone anabolic activity of the PDE‐inhibitor PTX, has an immunocentric basis, involving Wnt‐10b production by CD8+ T cells. When wild‐type (WT) mice were administered PTX, biochemical markers of both bone resorption and formation were significantly increased, with net bone gain in the axial skeleton, as quantified by micro‐computed tomography (μCT). By contrast, PTX increased only bone resorption in T cell knockout (KO) mice, causing net bone loss. Reconstituting T cell–deficient mice with WT, but not Wnt‐10b knockout (KO) CD8+ T cells, rescued bone formation and prevented bone loss. To study the role of cAMP signaling in Wnt‐10b expression, reverse‐transcription polymerase chain reaction (RT‐PCR) and luciferase‐reporter assays were performed using primary T cells. PDE inhibitors intensified Wnt‐10b promoter activity and messenger RNA (mRNA) accumulation in CD3 and CD28 activated CD8+ T cells. In contrast, inhibiting the cAMP pathway mediators protein kinase A (PKA) and cAMP response element‐binding protein (CREB), suppressed Wnt‐10b expression by T cells activated in the absence of CD28 co‐stimulation. In conclusion, the data demonstrate a key role for Wnt‐10b production by CD8+ T cells in the bone anabolic response to PDE‐inhibitors and reveal competing T cell–independent pro‐resorptive properties of PTX, which dominate under T cell–deficient conditions. Selective targeting of CD8+ T cells by PDE inhibitors may be a beneficial approach for promoting bone regeneration in osteoporotic conditions. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Joint Transcriptome and Metabolome Analysis Prevails the Biological Mechanisms Underlying the Pro-Survival Fight in In Vitro Heat-Stressed Granulosa Cells.

Simple SummaryThis study integrates the differentially expressed genes and important metabolites found in the transcriptome and metabolome analyses of in vitro acute heat-stressed bovine granulosa cells. Proteins and vitamin B catabolism were the most; tryptophan, proline, pyridoxine, and riboflavin (downregulated metabolites in heat stress) were enriched in many joint pathways. Genes AOX1, PYGM, NOS2, and SLC16A3 (all upregulated metabolites in heat stress) were found to have central roles in metabolic changes in response to heat stress. The negative effects of acute heat stress can be attributed to oxidative stress-driven changes at the inflammation metabolism nexus.Previous studies reported the physical, transcriptome, and metabolome changes in in vitro acute heat-stressed (38 °C versus 43 °C for 2 h) bovine granulosa cells. Granulosa cells exhibited transient proliferation senescence, oxidative stress, an increased rate of apoptosis, and a decline in steroidogenic activity. In this study, we performed a joint integration and network analysis of metabolomic and transcriptomic data to further narrow down and elucidate the role of differentially expressed genes, important metabolites, and relevant cellular and metabolic pathways in acute heat-stressed granulosa cells. Among the significant (raw p-value < 0.05) metabolic pathways where metabolites and genes converged, this study found vitamin B6 metabolism, glycine, serine and threonine metabolism, phenylalanine metabolism, arginine biosynthesis, tryptophan metabolism, arginine and proline metabolism, histidine metabolism, and glyoxylate and dicarboxylate metabolism. Important significant convergent biological pathways included ABC transporters and protein digestion and absorption, while functional signaling pathways included cAMP, mTOR, and AMPK signaling pathways together with the ovarian steroidogenesis pathway. Among the cancer pathways, the most important pathway was the central carbon metabolism in cancer. Through multiple analysis queries, progesterone, serotonin, citric acid, pyridoxal, L-lysine, succinic acid, L-glutamine, L-leucine, L-threonine, L-tyrosine, vitamin B6, choline, and CYP1B1, MAOB, VEGFA, WNT11, AOX1, ADCY2, ICAM1, PYGM, SLC2A4, SLC16A3, HSD11B2, and NOS2 appeared to be important enriched metabolites and genes, respectively. These genes, metabolites, and metabolic, cellular, and cell signaling pathways comprehensively elucidate the mechanisms underlying the intricate fight between death and survival in acute heat-stressed bovine granulosa cells and essentially help further our understanding (and will help the future quest) of research in this direction.

Granulosa cell transcriptomic study reveals the differential regulation of lncRNAs and mRNAs related to follicle development in goat

Mammalian follicle development is a complex biological process regulated by several factors. More than 99% of the follicles in goat ovaries will be atresia and only a few will eventually mature and ovulate. To investigate the potential long non-coding RNAs (lncRNAs) that regulate the expression of genes associated with follicular dominance or atresia, RNA-seq was performed on dominant follicles (DFs) and subordinate follicles (SFs) of granulosa cells from goats at the first follicular wave. A total of 92 differentially expressed lncRNAs and 676 differentially expressed mRNAs were detected in both types of follicles. The qRT-PCR results were consistent with the transcriptome sequencing data. Kyoto Encyclopedia of Genes and Genomes analysis of the differentially expressed mRNAs revealed that LHR and LDLR are associated with follicle dominance and are involved in the ovarian steroidogenesis pathway. The co-located mRNAs CALM2 and PPP1CA were significantly enriched during oocyte meiosis and in the cAMP and oxytocin signalling pathways. The co-expressed mRNAs were significantly enriched in the oestrogen signalling pathway and in ovarian steroidogenesis and progesterone-mediated oocyte maturation. A co-expression network of lncRNAs, target genes and differentially expressed genes was constructed. Follicle development-related genes, such as LDLR, NOTCH1 and FGF12, were included. These findings expand the lncRNA catalogue and provide a basis for further studies on the mechanism of regulating follicular development in goats.

The Expression Profile, Clinical Application and Potential Tumor Suppressing Mechanism of hsa_circ_0001675 in Head and Neck Carcinoma.

PurposeThis study sought to identify circular RNAs (circRNA) that participate in the regulation of head and neck cancer (HNC), analyze their clinical application, and predict their molecular mechanism during HNC.Materials and MethodsHigh-throughput sequencing was used to analyze circRNA expression in 18 matched HNC and adjacent normal tissues. Target circRNAs with significantly differential expression were obtained. In 103 HNC and adjacent normal tissues, real-time fluorescent quantitative PCR (qRT-PCR) was used to verify the differential expression of target circRNAs. This data was combined with clinicopathological information to analyze the diagnostic value of target circRNA. Bioinformatics was used to find target circRNAs that acted as competitive endogenous RNA (ceRNA) and construct a circRNA-miRNA-mRNA regulatory network. mRNA expression was verified by immunohistochemistry (IHC).ResultsA total of 714 differentially expressed circRNAs were detected in HNC, and the low expression of hsa_circ_0001675 was particularly significant (fold change [FC] = -4.85, P = 6.305E-05). hsa_circ_0001675 had significantly lower expression in HNC than in normal tissue (P < 0.01). Low hsa_circ_0001675 expression was positively associated with tumor invasion and clinical staging (P < 0.05), and its area under the ROC curve (AUC) was 0.7776. Low hsa_circ_0001675 expression also correlated with the overall survival (OS) rate and the progression-free survival (PFS) rate of HNC patients (P < 0.001). Bioinformatics was used to construct a ceRNA network of hsa_circ_0001675 with six differentially expressed miRNAs (hsa-miR-330-5p, hsa-miR-498, hsa-miR-532-3p, hsa-miR-577, hsa-miR-1248, and hsa-miR-1305) and 411 differentially expressed mRNAs and found that the neuroactive ligand-receptor interaction, and the cAMP and calcium signaling pathways were particularly enriched. Further bioinformatics and IHC analysis showed that miR577/TESC is the likely downstream signaling pathway for hsa_circ_0001675.ConclusionThis study showed that hsa_circ_0001675 is downregulated in HNC and could be an effective biomarker for HNC diagnosis. In addition, hsa_circ_0001675 may have a potential ceRNA mechanism and suppress HNC disease progression through the hsa_circ_0001675-miRNA-mRNA axis.

Potential Therapeutic Effects of Long-Term Stem Cell Administration: Impact on the Gene Profile and Kidney Function of PKD/Mhm (Cy/+) Rats.

Cystic kidney disease (CKD) is a heterogeneous group of genetic disorders and one of the most common causes of end-stage renal disease. Here, we investigate the potential effects of long-term human stem cell treatment on kidney function and the gene expression profile of PKD/Mhm (Cy/+) rats. Human adipose-derived stromal cells (ASC) and human skin-derived ABCB5+ stromal cells (2 × 106) were infused intravenously or intraperitoneally monthly, over 6 months. Additionally, ASC and ABCB5+-derived conditioned media were administrated intraperitoneally. The gene expression profile results showed a significant reprogramming of metabolism-related pathways along with downregulation of the cAMP, NF-kB and apoptosis pathways. During the experimental period, we measured the principal renal parameters as well as renal function using an innovative non-invasive transcutaneous device. All together, these analyses show a moderate amelioration of renal function in the ABCB5+ and ASC-treated groups. Additionally, ABCB5+ and ASC-derived conditioned media treatments lead to milder but still promising improvements. Even though further analyses have to be performed, the preliminary results obtained in this study can lay the foundations for a novel therapeutic approach with the application of cell-based therapy in CKD.

Role of Teneurin C-Terminal Associated Peptides (TCAP) on Intercellular Adhesion and Communication.

The teneurin C-terminal associated peptides (TCAP) are encoded by the terminal exon of all metazoan teneurin genes. Evidence supports the liberation of a soluble TCAP peptide either by proteolytic cleavage from the mature transmembrane teneurin protein or by a separately transcribed mRNA. Synthetic versions of TCAP, based on its genomic structure, are efficacious at regulating intercellular communication by promoting neurite outgrowth and increasing dendritic spine density in vitro and in vivo in rodent models. This is achieved through cytoskeletal re-arrangement and metabolic upregulation. The putative receptors for TCAPs are the latrophilin (LPHN) family of adhesion G-protein coupled receptors, which facilitate TCAP’s actions through G-proteins associated with cAMP and calcium-regulating signalling pathways. The teneurin/TCAP and latrophilin genes are phylogenetically ancient, likely serving primitive functions in cell adhesion and energy regulation which have been since adapted for a more complex role in synaptogenesis in vertebrate nervous systems.

Loss-of-function variants in the schizophrenia risk gene SETD1A alter neuronal network activity in human neurons through the cAMP/PKA pathway.

Heterozygous loss-of-function (LoF) mutations in SETD1A, which encodes a subunit of histone H3 lysine 4 methyltransferase, cause a neurodevelopmental syndrome and increase the risk for schizophrenia. Using CRISPR-Cas9, we generate excitatory/inhibitory neuronal networks from human induced pluripotent stem cells with a SETD1A heterozygous LoF mutation (SETD1A+/-). Our data show that SETD1A haploinsufficiency results in morphologically increased dendritic complexity and functionally increased bursting activity. This network phenotype is primarily driven by SETD1A haploinsufficiency in glutamatergic neurons. In accordance with the functional changes, transcriptomic profiling reveals perturbations in gene sets associated with glutamatergic synaptic function. At the molecular level, we identify specific changes in the cyclic AMP (cAMP)/Protein Kinase A pathway pointing toward a hyperactive cAMP pathway in SETD1A+/- neurons. Finally, by pharmacologically targeting the cAMP pathway, we are able to rescue the network deficits in SETD1A+/- cultures. Our results demonstrate a link between SETD1A and the cAMP-dependent pathway in human neurons.

BS-400-08 HETEROGENOUS CAMP SIGNALING IN THE INTACT HEART IS SEX DEPENDENT

cAMP is key for transducing autonomic signals into downstream electrophysiological responses. Previous studies have shown intracellular heterogeneity and compartmentalization of cAMP signaling. Yet, if cAMP signaling occurs heterogeneously throughout the intact heart, and how this translates into functional responses, has not been explored. To determine the spatiotemporal kinetics of cAMP signaling in the intact heart and the underlying mechanisms responsible. Male and female cardiac-specific CAMPER reporter mice that report cAMP binding by changes in FRET were used at 12 weeks. Hearts were excised and Langendorff-perfused for simultaneous cAMP and Vm imaging on a novel integrated whole heart optical imaging system. Following perfusion, tissue samples from base and apex were assessed for phosphodiesterase (PDE) activity. In male hearts, cAMP was uniformly activated in response to β-AR stimulation with bolus norepinephrine (NE, 1.5 μM). Conversely, in female hearts NE led to a greater change in cAMP activity in basal regions vs. the apex (n=7, p<0.05). Moreover, cAMP deactivation was slower in the base vs. apex, in female (n=6, p<0.01) but not male hearts (n=6). Apex-base differences were also evident following PDE inhibition with IBMX (100 μM), with a greater change in cAMP activity in the apex vs. base in both female (n=7, p<0.001) and male hearts (n=5, p<0.05). Likewise, PDE activity assays showed higher total PDE activity in apical regions (n=10, p<0.01), with more apical PDE activity in female vs. male hearts (n=5, p<0.05). In female hearts, faster apical cAMP deactivation following bolus NE was associated with a significant difference in action potential duration (APD80) between apex and base (n=3, p<0.05), but APD80 was not significantly different between regions in male hearts. Using novel whole heart imaging, we have shown female hearts display lower maximal cAMP activity and faster deactivation in the apex, in part, due to elevated PDE activity in this region. This heterogeneity was not observed in male hearts. These findings may have important implications for electrophysiological responses regulated by the cAMP pathway, particularly in heart failure, where PDE activity is altered.

Hyperpolarization‐Activated Current Shows Excitatory Effect on Fictive Locomotion in the Neonatal Mouse Under Hyperdopaminergic Conditions

Restless Leg Syndrome (RLS) is a chronic sensorimotor disorder that has recently been linked to a downregulation in adenosine A1 receptors (A1R), leading to decreased formation of adenosine‐dopamine heteromers and a disinhibited or hyperdopaminergic transmission pathway within the central nervous system. Dopamine D1 receptor (D1R) activation has been shown to modulate HCN1 channels via its stimulatory effect on cAMP production to increase the hyperpolarization‐activated cation current (Ih). Ih has been related to the regulation of bursting activity in rhythmic motor networks, specifically in controlling the firing frequency of the system. Our study aims to establish the importance of Ih in mammalian locomotion under hyperdopaminergic conditions mimicking those seen in RLS patients. We hypothesized that blocking Ih would increase the cycle period and destabilize the rhythm.MethodsWe isolated the spinal cords of neonatal (P0‐P5; n = 6) Swiss Webster mice via ventral laminectomy and recorded the extracellular activity of ventral roots L2 and L5 using suction electrodes to assess burst amplitude, burst duration, and cycle period. Fictive locomotion was induced using serotonin (5HT; 9‐15μM) and N‐methyl‐D‐aspartate (NMDA; 6 μM) before adding dopamine (DA; 50μM) to determine the effect of DA on the system. We then used ZD7288 (1μM) block HCN1 channels to investigate the role of Ih in motor‐like activity.ResultsBlocking Ih caused a significant increase in all three parameters relative to the 5HT/NMDA control in the L2 roots, but only cycle period was significantly increased relative to the high dopamine condition. There were no significant changes in any of the parameters in the L5 roots.ConclusionsOur results suggest that Ih primarily plays an excitatory role in locomotor regulation under hyperdopaminergic conditions, such as those seen in RLS. The differential effects between roots may suggest that one is more susceptible to the blockade of Ih by ZD7288; however, given the large degree of variability in the L5 data, a larger sample size is needed to confirm this hypothesis. Future experiments include collecting more data on L5 to confirm if there are differential effects, as well as pharmacological approaches to confirm that D1R is upregulating Ih via the cAMP pathway.

Effects of RTI‐567 on the Neuropeptide S System

Anxiety and post‐traumatic stress have increasingly become more prominent disorders and options for treatment remain limited. One of the most prescribed therapeutics are benzodiazepines. Unfortunately, these anxiolytics have negative side effects and are not as effective in the long term. The recently discovered Neuropeptide S (NPS) system may lead to an alternative, potentially improved, line of therapeutics. This system consists of a known endogenous neuropeptide, Neuropeptide S and a G‐protein‐coupled receptor termed the Neuropeptide S receptor (NPSR). Activation of this receptor leads to an increase in intracellular calcium and a mobilization of cAMP. This is associated with behavioral phenotypes such as anxiolysis, enhanced memory consolidation, and hyperlocomotion. The NPSR is highly expressed in the amygdala linking the NPS system with fear and anxiety. Previously, as part of our ongoing drug discovery program, a biased agonist (RTI‐263) was identified that preferentially enhances the calcium signaling pathway over the cAMP pathway following NPSR binding. This biased agonist also reduced hyperlocomotion or arousal observed with NPS, while maintaining the anxiolytic component. In the present study we test another compound (RTI‐567) using classic anxiolytic, locomotor, and memory testing paradigms such as the light dark box, open field, and inhibitory avoidance. Our overall goal is to identify novel synthetic agonists with reduced side effects and improved long‐term efficacy.

5HTR1E receptor interacts with Neurotrophic factor‐α1 and serotonin to activate two distinct signaling pathways

Neurotrophic factor‐α1/Carboxypeptidase E (NFα1/CPE) is a prohormone processing enzyme intracellularly and as a neurotrophic factor, extracellularly. Previously we showed that extracellular treatment of hippocampal neurons with NFα1/CPE protected them from oxidative stress‐induced cell death through ERK/BCL2 signaling. Based on these results, we hypothesized that NFα1/CPE activation of the ERK/BCL2 pathway to mediate neuroprotection should involve interaction with receptors at the cell membrane. We have identified 5‐hydroxytryptamine receptor 1E (5HTR1E), a Gi coupled serotonin receptor, as an extracellular binding partner of NFα1/CPE. This interaction was confirmed by co‐IP and pull‐down experiments followed by radioligand binding studies in HEK293‐5HTR1E overexpressing cells. Molecular modeling studies also predicted that NFα1/CPE interacted with HTR1E, not at the serotonin binding pocket, but via the extracellular loop 2 (ECL2) of 5HTR1E, forming 3 stable salt bridges with specific amino acid residues in NFα1/CPE. To identify the HTR1E downstream signaling pathways, we used HEK293 cells stably expressing 5HTR1E. First, we tested the dose dependent effect of both serotonin and NFα1/CPE on ERK, and cAMP signaling and found that both can increase ERK phosphorylation while cAMP pathway activation is serotonin dependent only. To further dissect the 5HTR1E mediated functions of both serotonin and NFα1/CPE we tested Gi, Gq, PKA, and PI3K inhibitors and observed that these proteins are not involved in NFα1/CPE mediated ERK phosphorylation, while serotonin‐stimulated ERK activation was Gi‐PKA dependent. We then explored the involvement of β‐arrestin, an alternative coupling mechanism with GPCRs for ERK activation. Studies in 5HTR1E expressing‐HEK293‐β‐arrestin KO cells revealed that NFα1/CPE‐5HTR1E complex was unable to induce ERK signaling in the absence of β‐arrestin proteins. Furthermore, molecular docking studies revealed a very strong coupling of b‐arrestin to intracellular loop (ICL) 2 and ICL3 of HTR1E. Using LDH cytotoxicity assay we demonstrated that NF‐α1/CPE was able to protect HEK‐293‐5HTR1E expressing cells against H2O2‐induced cytotoxicity, via increasing pro‐survival protein BCL2 expression; however, this protective effect of NFα1/CPE was diminished in the β‐arrestin KO cells. Our study identified two ligands for HTR1E: NFα1/CPE which interacts with HTR1E to activate β‐arrestin/ERK/BCL2 signaling pathway for protecting cells against oxidative stress; and serotonin which activated a Gi‐cAMP‐dependent pathway, the physiological role is under investigation.

A Stressful Question ‐ How Does 3′ 5′ cyclic adenosine monophosphate Signaling Affect Oxygen Metabolism Within Malignant Hyperthermia Susceptible (MHS) B‐lymphocytes?

BackgroundEven though the MHS trait is inherited by an autosomal dominant pattern, the variable penetrance and symptom presentation suggest involvement of oligogenic or post‐translational modifications. Since malignant hyperthermia (MH) includes hypermetabolic pathologies as its defining characteristics, the metabolic pathways offer biomarker targets which may segregate with the MHS phenotype.ProblemIdentifying the malignant hyperthermia susceptible (MHS) trait using pathogenic mutations has limitations. Causative mutations within the ryanodine receptor type I (RYR1),the SH3 and cysteine rich domain 3 (STAC3),or the (CACNA1S), voltage dependent calcium channel, alpha‐1s subunit genes account for only 50‐86% of subjects diagnosed with MH.HypothesisThe adrenergic agonist norepinephrine (NOR) and the membrane permeable 3′,5′ cyclic adenosine monophosphate (cAMP) analog, adenosine 3ʹ,5ʹ‐cyclic monophosphate, 8‐bromo‐, sodium salt, (8Br‐cAMP‐Na) will significantly alter oxygen metabolism between human MHS and MH non‐susceptible (MHN) b‐lymphocyte groups.MethodsMHN and MHS cells (8.5x104 and 3.7 x104 cells/well, respectively), were treated with NOR 1μM, or 10μM 8Br‐cAMP‐Na, for 1 hr. Electron transport chain inhibitors were sequentially added to form a bioenergetic profile. The Seahorse Xfe96 AnalyzerTM analyzed the oxygen consumption rate (OCR, pmol/min/cells) to calculate the basal and maximal OCR and the spare respiratory capacity (SRC). This value quantifies the metabolic ability of the cell to respond to an energetic demand.ResultsWhen compared to the naive MHN group, 8Br‐cAMP‐Na decreased the MHN SRC by 48%, p &lt;.001. In contrast, the SRC increased 67%, p&lt; .01, within the 8Br‐cAMP‐Na MHS group vs the MHS naive group. 1μM NOR produced similar SRC changes within each group. The SRC decreased by 22% ,p &lt;.05, and increased by 35%, p &gt;.05, within the NOR MHN and MHS groups, respectively (n = 4‐12 wells/group).ConclusionThe intracellular cAMP signaling affect the oxygen metabolism differently within MHN and MHS groups during adrenergic stress. The cAMP pathways contributing to a subclinical hypermetabolic response need further research. The adrenergic signaling may “prime” the conditions for an MH crisis until a specific metabolic threshold is obtained. The MHN group compensates for the increased metabolism and does not reach a thresh hold.DisclaimersThe opinions and assertions expressed herein are those of the author(s) and do not necessarily reflect official policy or position of the Uniformed Services University or the Department of Defense.This work was prepared by a military or civilian employee of the US Government as part of the individual’s official duties and therefore is in the public domain and does not possess copyright protection (public domain information may be freely distributed and copied; however, as a courtesy it is requested that the Uniformed Services University and the authors be given an appropriate acknowledgement).The protocol DBS.2019.008, was reviewed and approved by the USU Institutional Review Board, in accordance with all applicable Federal regulations governing the protection of humans in research.

Role of the Cytoskeleton in Steroidogenesis.

Steroidogenesis in the adrenal cortex or gonads is a complicated process modulated by various elements either at the tissue or molecular level. The substrate cholesterol is first delivered to the outer membrane of mitochondria, undergoing a series of enzymatic reactions along with the material exchange between the mitochondria and the ER (endoplasmic reticulum) and ultimately yielding various steroids, such as aldosterone, cortisol, testosterone, and estrone. Several valves are set to adjust the amount of production as per the needs, e.g., StAR (steroidogenic acute regulator) controls the traffic of cholesterol from the outer membrane to the inner membrane of mitochondria which is a rate-limiting step. Moreover, the "need" is partly reflected by trophic signals, like ACTH, LH, and downstream pathways, such as the intracellular cAMP pathway, representing the endocrinal regulation of steroid synthesis. The coordinated activities of these related factors are all associated with another crucial cellular constituent, the cytoskeleton, which plays a crucial role in cellular architecture and substrate trafficking. Though considerable studies have been performed regarding steroid synthesis, details regarding the upstream signaling pathways and mechanisms of the regulation by the cytoskeleton network still remain unclear. The metabolism and interplays of the pivotal cellular organelles with cytoskeleton are worth exploring as well. This review summarizes the research of different periods, describing the roles of specific cytoskeleton elements in steroidogenesis and related signaling pathways involved in steroid synthesis. In addition, we discuss the inner cytoskeletal network involved in steroidogenic processes, such as mitochondrial movement, organelle interactions, and cholesterol trafficking.

Identification and characterization of circular RNAs in Longissimus dorsi muscle tissue from two goat breeds using RNA-Seq.

Circular RNAs (circRNAs) are a class of non-coding RNA that play crucial roles in the growth and development of skeletal muscle. However, little is known about the role of circRNAs in caprine skeletal muscle. In this study, the size of muscle fiber and the expression profiles of circRNAs were compared in Longissimus dorsi muscle of Liaoning cashmere (LC) goats and Ziwuling black (ZB) goats with significant phenotypic differences in meat production performance, using hematoxylin and eosin staining and RNA-Seq, respectively. The size of muscle fiber in LC goats was larger than those in ZB goats (P < 0.05). A total of 10,875 circRNAs were identified and 214 of these were differentially expressed between the two caprine breeds. The parent genes of differentially expressed circRNAs were mainly enriched in connective tissue development, Rap1, cGMP-PKG, cAMP and Ras signaling pathway. In conclusion, circRNAs may play important roles in skeletal mass, meat production performance and meat quality traits in goats. The results provide an improved understanding of the functions of circRNAs in skeletal muscle development of goats.

Characteristics of the MicroRNA Expression Profile of Exosomes Released by Vero Cells Infected with Porcine Epidemic Diarrhea Virus.

Exosomes are nanoscale vesicles actively secreted by a variety of cells. They contain regulated microRNA (miRNA), allowing them to function in intercellular communication. In the present study, the role of exosomal miRNAs in porcine epidemic diarrhea virus (PEDV) infection was investigated using exosomes isolated from Vero cells infected with PEDV. The results of transmission electron microscopy observation showed that the exosomes are spherical in shape, uniform in size, and negatively stained in the membrane. Nanoparticle tracking analysis showed that the average exosome particle size is 130.5 nm. The results of miRNA sequencing showed that, compared with the control group, a total of 115 miRNAs are abnormally expressed in the exosomes of infected cells. Of these, 80 miRNAs are significantly upregulated and 35 miRNAs are significantly downregulated. Functional annotation analysis showed that the differentially expressed miRNAs are associated with PEDV infection through interaction with the cAMP, Hippo, TGF-beta, HIF-1, FoxO, MAPK, and Ras signaling pathways. Thus, our findings provide important information about the effects of PEDV infection on exosomal miRNA expression and will aid the search for potential anti-PEDV drug candidates.

Proteomic Analysis of the Role of the Adenylyl Cyclase-cAMP Pathway in Red Blood Cell Mechanical Responses.

Red blood cell (RBC) deformability is modulated by the phosphorylation status of the cytoskeletal proteins that regulate the interactions of integral transmembrane complexes. Proteomic studies have revealed that receptor-related signaling molecules and regulatory proteins involved in signaling cascades are present in RBCs. In this study, we investigated the roles of the cAMP signaling mechanism in modulating shear-induced RBC deformability and examined changes in the phosphorylation of the RBC proteome. We implemented the inhibitors of adenylyl cyclase (SQ22536), protein kinase A (H89), and phosphodiesterase (PDE) (pentoxifylline) to whole blood samples, applied 5 Pa shear stress (SS) for 300 s with a capillary tubing system, and evaluated RBC deformability using a LORRCA MaxSis. The inhibition of signaling molecules significantly deteriorated shear-induced RBC deformability (p < 0.05). Capillary SS slightly increased the phosphorylation of RBC cytoskeletal proteins. Tyrosine phosphorylation was significantly elevated by the modulation of the cAMP/PKA pathway (p < 0.05), while serine phosphorylation significantly decreased as a result of the inhibition of PDE (p < 0.05). AC is the core element of this signaling pathway, and PDE works as a negative feedback mechanism that could have potential roles in SS-induced RBC deformability. The cAMP/PKA pathway could regulate RBC deformability during capillary transit by triggering significant alterations in the phosphorylation state of RBCs.