cAMP-PKA/EPAC signaling and cancer: the interplay in tumor microenvironment.

Cancer is a complex disease resulting from abnormal cell growth that is induced by a number of genetic and environmental factors. The tumor microenvironment (TME), which involves extracellular matrix, cancer-associated fibroblasts (CAF), tumor-infiltrating immune cells and angiogenesis, plays a critical role in tumor progression. Cyclic adenosine monophosphate (cAMP) is a second messenger that has pleiotropic effects on the TME. The downstream effectors of cAMP include cAMP-dependent protein kinase (PKA), exchange protein activated by cAMP (EPAC) and ion channels. While cAMP can activate PKA or EPAC and promote cancer cell growth, it can also inhibit cell proliferation and survival in context- and cancer type-dependent manner. Tumor-associated stromal cells, such as CAF and immune cells, can release cytokines and growth factors that either stimulate or inhibit cAMP production within the TME. Recent studies have shown that targeting cAMP signaling in the TME has therapeutic benefits in cancer. Small-molecule agents that inhibit adenylate cyclase and PKA have been shown to inhibit tumor growth. In addition, cAMP-elevating agents, such as forskolin, can not only induce cancer cell death, but also directly inhibit cell proliferation in some cancer types. In this review, we summarize current understanding of cAMP signaling in cancer biology and immunology and discuss the basis for its context-dependent dual role in oncogenesis. Understanding the precise mechanisms by which cAMP and the TME interact in cancer will be critical for the development of effective therapies. Future studies aimed at investigating the cAMP-cancer axis and its regulation in the TME may provide new insights into the underlying mechanisms of tumorigenesis and lead to the development of novel therapeutic strategies.

N-acetyl cysteine turns EPAC activators into potent killers of acute lymphoblastic leukemia cells.

Today, the majority of patients with pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL, hereafter ALL) survive their disease, but many of the survivors suffer from life-limiting late effects of the treatment. ALL develops in the bone marrow, where the cells are exposed to cAMP-generating prostaglandin E2. We have previously identified the cAMP signaling pathway as a putative target for improved efficacy of ALL treatment, based on the ability of cAMP signaling to reduce apoptosis induced by DNA damaging agents. In the present study, we have identified the antioxidant N-acetyl cysteine (NAC) as a powerful modifier of critical events downstream of the cell-permeable cAMP analog 8-(4-chlorophenylthio) adenosine-3', 5'- cyclic monophosphate (8-CPT). Accordingly, we found NAC to turn 8-CPT into a potent killer of ALL cells in vitro both in the presence and absence of DNA damaging treatment. Furthermore, we revealed that NAC in combination with 8-CPT is able to delay the progression of ALL in a xenograft model in NOD-scid IL2Rγnull mice. NAC was shown to rely on the ability of 8-CPT to activate the guanine-nucleotide exchange factor EPAC, and we demonstrated that the ALL cells are killed by apoptosis involving sustained elevated levels of calcium imposed by the combination of the two drugs. Taken together, we propose that 8-CPT in the presence of NAC might be utilized as a novel strategy for treating pediatric ALL patients, and that this powerful combination might be exploited to enhance the therapeutic index of current ALL targeting therapies.

Real-Time Spatiotemporal Measurement of Extracellular Signaling Molecules Using an Aptamer Switch-Conjugated Hydrogel Matrix.

Cells rely on secreted signaling molecules to coordinate essential biological functions including development, metabolism, and immunity. Unfortunately, such signaling processes remain difficult to measure with sufficient chemical specificity and temporal resolution. To address this need, an aptamer-conjugated hydrogel matrix that enables continuous fluorescent measurement of specific secreted analytes - in two dimensions, in real-time is developed. As a proof of concept, real-time imaging of inter-cellular cyclic adenosine 3',5'-monophosphate (cAMP) signals in Dictyostelium discoideum amoeba cells is performed. A set of aptamer switches that generate a rapid and reversible change in fluorescence in response to cAMP signals is engineered. By combining multiple switches with different dynamic ranges, measure cAMP concentrations spanning three orders of magnitude in a single experiment can be measured. These sensors are embedded within a biocompatible hydrogel on which cells are cultured and their cAMP secretions can be imaged using fluorescent microscopy. Using this aptamer-hydrogel material system, the first direct measurements of oscillatory cAMP signaling that correlate closely with previous indirect measurements are achieved. Using different aptamer switches, this approach can be generalized for measuring other secreted molecules to directly visualize diverse extracellular signaling processes and the biological effects that they trigger in recipient cells.

Sirtuin 6 ameliorates arthritis through modulating cyclic AMP-responsive element binding protein/CCN1/cyclooxygenase 2 pathway in osteoblasts.

INTRODUCTION: CCN1 is an immediate-early gene product pivotal for arthritis progression. We have previously shown that sirtuin 6 (SIRT6) inhibited hypoxia-induced CCN1 expression in osteoblasts. Herein we examined the contribution of cyclic AMP-responsive element binding protein (CREB)/CRE to this suppressive action and the influence of CCN1 on cyclooxygenase (COX) 2 synthesis. MATERIALS AND METHODS: MC3T3-E1 murine osteoblasts were cultured under normoxia (21% oxygen) or hypoxia (2% oxygen). Expressions of CCN1, phospho-CREB (Ser133), COX2 and relevant kinases were assessed by Western blot. SIRT6 was overexpressed in cultured osteoblasts and arthritic joints by a lentiviral-based technique. Activities of CCN1 gene promoter constructs were examined by luciferase reporter assay. Interaction between CREB and CCN1 promoter was assessed by chromatin immunoprecipitation (ChIP). Collagen-induced arthritis (CIA) was established in 20 rats to evaluate the effects of SIRT6 therapy on osteoblastic expressions of phospho-CREB, CCN1 and COX2. RESULTS: SIRT6 suppressed hypoxia-enhanced CCN1 expression and CREB phosphorylation. Attenuation of calcium/calmodulin-dependent protein kinase II (CaMKII) may be responsible for SIRT6-induced CREB inhibition. CRE at - 286 bp upstream of the ATG start codon was essential for CCN1 expression under hypoxia and SIRT6 reduced hypoxia-stimulated CREB/CRE interaction. Forced expression of CREB rescued SIRT6-suppressed CCN1 synthesis. CCN1 induced COX2 expression in osteoblasts. In rat CIA, the therapeutic effect of SIRT6 was accompanied by decreases in osteoblastic expressions of phospho-CREB, CCN1 and COX2. CONCLUSION: Our study indicated that the benefits of SIRT6 to inflammatory arthritis and bone resorption are at least partially derived from its modulation of CREB/CCN1/COX2 pathway in osteoblasts.

Clinical pharmacology of cardiac cyclic AMP in human heart failure: too much or too little?

INTRODUCTION: Cyclic 3', 5'-adenosine monophosphate (cAMP) is a major signaling hub in cardiac physiology. Although cAMP signaling has been extensively studied in cardiac cells and animal models of heart failure (HF), not much is known about its actual amount present inside human failing or non-failing cardiomyocytes. Since many drugs used in HF work via cAMP, it is crucial to determine the status of its intracellular levels in failing vs. normal human hearts. AREAS COVERED: Only studies performed on explanted/excised cardiac tissues from patients were examined. Studies that contained no data from human hearts or no data on cAMP levels per se were excluded from this perspective's analysis. EXPERT OPINION: Currently, there is no consensus on the status of cAMP levels in human failing vs. non-failing hearts. Several studies on animal models may suggest maladaptive (e.g. pro-apoptotic) effects of cAMP on HF, advocating for cAMP lowering for therapy, but human studies almost universally indicate that myocardial cAMP levels are deficient in human failing hearts. It is the expert opinion of this perspective that intracellular cAMP levels are too low in human failing hearts, contributing to the disease. Strategies to increase (restore), not decrease, these levels should be pursued in human HF.

Noncanonical Regulation of cAMP-Dependent Insulin Secretion and Its Implications in Type 2 Diabetes.

Impaired glucose tolerance (IGT) and ß-cell dysfunction in insulin resistance associated with obesity lead to type 2 diabetes (T2D). Glucose-stimulated insulin secretion (GSIS) from ß-cells occurs via a canonical pathway that involves glucose metabolism, ATP generation, inactivation of K ATP channels, plasma membrane depolarization, and increases in cytosolic concentrations of [Ca 2+ ] c . However, optimal insulin secretion requires amplification of GSIS by increases in cyclic adenosine monophosphate (cAMP) signaling. The cAMP effectors protein kinase A (PKA) and exchange factor activated by cyclic-AMP (Epac) regulate membrane depolarization, gene expression, and trafficking and fusion of insulin granules to the plasma membrane for amplifying GSIS. The widely recognized lipid signaling generated within ß-cells by the ß-isoform of Ca 2+ -independent phospholipase A 2 enzyme (iPLA 2 ß) participates in cAMP-stimulated insulin secretion (cSIS). Recent work has identified the role of a G-protein coupled receptor (GPCR) activated signaling by the complement 1q like-3 (C1ql3) secreted protein in inhibiting cSIS. In the IGT state, cSIS is attenuated, and the ß-cell function is reduced. Interestingly, while ß-cell-specific deletion of iPLA 2 ß reduces cAMP-mediated amplification of GSIS, the loss of iPLA 2 ß in macrophages (MØ) confers protection against the development of glucose intolerance associated with diet-induced obesity (DIO). In this article, we discuss canonical (glucose and cAMP) and novel noncanonical (iPLA 2 ß and C1ql3) pathways and how they may affect ß-cell (dys)function in the context of impaired glucose intolerance associated with obesity and T2D. In conclusion, we provide a perspective that in IGT states, targeting noncanonical pathways along with canonical pathways could be a more comprehensive approach for restoring ß-cell function in T2D. © 2023 American Physiological Society. Compr Physiol 13:5023-5049, 2023.

Caffeine reduces oxidative stress to protect against hyperoxia-induced lung injury via the adenosine A2A receptor/cAMP/PKA/Src/ERK1/2/p38MAPK pathway.

Objectives: Caffeine has been shown to reduce the incidence of bronchopulmonary dysplasia (BPD). To investigate the protective mechanism of caffeine in a hyperoxia-based cell model of BPD in vitro.Methods: Type II alveolar epithelial cells (AECs II) were isolated and randomly divided into 6 groups: the normal, hyperoxia, caffeine (50 µM caffeine), antagonist (5 µM ZM241385), agonist (5 µM CGS21680), and DMSO groups. Transfection with siRNA against adenosine A2A receptor (siA2AR) was performed in AECs II.Results: Caffeine alone or in combination with adenosine A2A receptor (A2AR) antagonist inhibited apoptosis, promoted proliferation and reduced oxidative stress (OS). The cyclic adenosine monophosphate (cAMP), protein kinase A (PKA) mRNA, A2AR mRNA and the protein levels of A2AR, phospho-Src, phospho-ERK1/2, phospho-P38 and cleaved caspase-3 were decreased in the caffeine and antagonist groups compared with that in the hyperoxia group. However, the effects of caffeine above were weakened by the A2AR agonist. Knockdown of A2AR showed similar results to caffeine.Discussion: Caffeine can reduce apoptosis, promote proliferation, and alleviate OS in hyperoxia-induced AECs II injury by inhibiting the A2AR/cAMP/PKA/Src/ERK1/2/p38MAPK signaling pathway. Caffeine and A2AR may serve as a promising therapeutic target for BPD in prematurity.

Polyvinyl alcohol, but not bovine serum albumin, promotes the induction of full-type hyperactivation in boar cyclic AMP analog-treated spermatozoa.

This study aimed to verify the effects of polyvinyl alcohol (PVA) and bovine serum albumin (BSA) on the induction of full-type hyperactivation in boar spermatozoa treated with a cyclic AMP analog (cBiMPS). Washed spermatozoa were treated with cBiMPS (100 µM) for 180 min. As shown in the assessment of sperm motility, PVA (0.05%-0.4%) significantly promoted the induction of full-type hyperactivation, whereas BSA (0.025%-0.4%) did not affect the induction. In comparative experiments, BSA (0.4%) effectively promoted the induction of full-type hyperactivation in bovine spermatozoa treated with cBiMPS, calyculin A (a protein phosphatase inhibitor), and digoxin (a Na+ /K+ -ATPase inhibitor), while PVA (0.1%) did not affect the induction. Western blotting showed that protein tyrosine phosphorylation states of >50 kDa sperm proteins were effectively enhanced by treatment with cBiMPS in the PVA/BSA-free medium and not affected by the addition of PVA (0.1%). The assessment of plasma membrane integrity indicated that BSA (0.4%) significantly decreased spermatozoa with intact plasma membranes. These results indicate that PVA (0.1%) promotes the induction of full-type hyperactivation and does not influence the protein tyrosine phosphorylation states in boar cBiMPS-treated spermatozoa. They also suggest that BSA should not be added to medium containing cBiMPS for boar spermatozoa.

Small Hepatitis B Virus Surface Antigen Promotes Hepatic Gluconeogenesis via Enhancing Glucagon/cAMP/Protein Kinase A/CREB Signaling.

Hepatitis B virus (HBV) is a major risk factor for serious liver diseases. The liver plays a unique role in controlling carbohydrate metabolism to maintain the glucose level within the normal range. Chronic HBV infection has been reported to associate with a high prevalence of diabetes. However, the detailed molecular mechanism underlying the potential association remains largely unknown. Here, we report that liver-targeted delivery of small HBV surface antigen (SHBs), the most abundant viral protein of HBV, could elevate blood glucose levels and impair glucose and insulin tolerance in mice by promoting hepatic gluconeogenesis. Hepatocytes with SHB expression also exhibited increased glucose production and expression of gluconeogenic genes glucose-6-phosphatase (G6pc) and phosphoenolpyruvate carboxykinase (PEPCK) in response to glucagon stimulation. Mechanistically, SHBs increased cellular levels of cyclic AMP (cAMP) and consequently activated protein kinase A (PKA) and its downstream effector cAMP-responsive element binding protein (CREB). SHBs-induced activation of CREB enhanced transcripts of gluconeogenic genes, thus promoting hepatic gluconeogenesis. The elevated cAMP level resulted from increased transcription activity and expression of adenylyl cyclase 1 (AC1) by SHBs through a binary E-box factor binding site (BEF). Taken together, we unveiled a novel pathogenic role and mechanism of SHBs in hepatic gluconeogenesis, and these results might highlight a potential target for preventive and therapeutic intervention in the development and progression of HBV-associated diabetes. IMPORTANCE Chronic HBV infection causes progressive liver damage and is found to be a risk factor for diabetes. However, the mechanism in the regulation of glucose metabolism by HBV remains to be established. In the current study, we demonstrate for the first time that the small hepatitis B virus surface antigen (SHBs) of HBV elevates AC1 transcription and expression to activate cAMP/PKA/CREB signaling and subsequently induces the expression of gluconeogenic genes and promotes hepatic gluconeogenesis both in vivo and in vitro. This study provides a direct link between HBV infection and diabetes and implicates that SHBs may represent a potential target for the treatment of HBV-induced metabolic disorders.

Cilostamide, a phosphodiesterase 3A inhibitor, sustains meiotic arrest of rat oocytes by modulating cyclic adenosine monophosphate level and the key regulators of maturation promoting factor.

Cilostamide, a phosphodiesterase 3A (Pde3A) inhibitor, is known to increase intraoocyte cyclic adenosine monophosphate (cAMP) level which is involved in sustaining meiotic arrest of the oocytes. To explore the mechanisms involved in the cilostamide-mediated meiotic arrest of the oocytes, the present study describes the effects of cilostamide on cAMP level and related factors involved in maturation of the oocytes at its different meiotic stages; diplotene, metaphase I (MI) and metaphase II (MII). The oocytes from these three stages were collected from rat ovary and incubated with 10 µM cilostamide for 3 h in CO2 incubator. The levels of cAMP, cyclic guanosine monophosphate (cGMP) and the key players of maintaining meiotic arrest during oocyte maturation; Emi2, Apc, Cyclin B1, and Cdk1, were analyzed in diplotene, MI and MII stages. Pde3A was found to be expressed at all three stages but with the lowest level in MI oocyte. As compared to the control sets, the cAMP concentration was found to be highest in MII whereas cGMP was highest in the diplotene stage of cilostamide-treated group. The treated group showed declined reactive oxygen species level as compared with the control counterparts. Relatively increased levels of the Emi2, Cyclin B1, and phosphorylated thr161 of Cdk1 versus declined levels of phosphorylated thr14/tyr15 of Cdk1 in diplotene and MII stage oocytes are known to be involved in maintaining meiotic arrest and all these factors were found to undergo similar pattern of change due to the treatment with cilostamide. The findings thus suggest that cilostamide treatment promotes meiotic arrest by Pde3A inhibition led increase of both cAMP and cGMP level vis-a-vis modulation of the related regulatory factors such as Emi2, CyclinB1, and phosphorylated status of Cdk1 in diplotene and MII stage oocytes. Such a mechanism of meiotic arrest could allow the oocyte to prepare itself for meiotic maturation and thereby to improve oocyte quality.

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

OBJECTIVES: Human 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 METHODS: Samples 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. RESULTS: After 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). CONCLUSION: Our 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.

Cyclic AMP and biofilms reveal the synergistic proliferation strategy of Pseudomonas aeruginosa and Escherichia coli under the costimulation of high concentrations of microplastics and enrofloxacin.

Microplastics (MPs) provide attachment sites for biofilm formation of microorganisms, which can promote their resistance to environmental stress has been proved. However, the effect of MPs on synergy survival among microorganisms under antibiotic stress remains unclear. In the present study, the proliferation of Escherichia coli and Pseudomonas aeruginosa was assessed under enrofloxacin stress with the influence of MPs. Here, MPs reduced the growth speed of E. coli and enhanced that of P. aeruginosa, especially at 12 h, but the final value of OD600 and CFU of both bacteria not be influenced. E. coli was enrofloxacin sensitive (MIC = 0.25 µg/mL), and a high MP concentration in the presence of enrofloxacin notably enhanced the biofilm formation ability of P. aeruginosa, but proliferation decreased. In the coculture system, the proliferation of E. coli (increased 1.42-fold) and P. aeruginosa (increased 1.06-fold) both increased under enrofloxacin stress (0.25 µg/mL) with high-concentration MP addition. P. aeruginosa may provide the biofilm matrix for E. coli to resist the stress of enrofloxacin. The high concentration of cyclic AMP secreted by E. coli may slightly inhibited biofilm formation, leading to a decrease in the fitness cost of P. aeruginosa; thus, the proliferation of P. aeruginosa increased. The present study is the first to show that MP combined with antibiotics stimulates the metabolic cooperation of bacteria to promote proliferation.

PGE2 inhibits neutrophil phagocytosis through the EP2R-cAMP-PTEN pathway.

Prostaglandin E2 (PGE2) is a potent lipid mediator of inflammation that modulates immune cell function by binding to unique G protein-coupled receptors (EP receptors). PGE2 production increases during microbial infection and inflammation. In this study, we assessed the effect of PGE2 on the phagocytosis of bacteria by neutrophils, which are key players during infection and inflammation. We also looked for specific EP receptor signaling pathways that contributed to the neutrophil phagocytic activity. PGE2 (50-1000 ng/ml) inhibited the phagocytosis of Escherichia coli by HL-60 human neutrophils in a concentration-dependent manner. Inhibition of neutrophil phagocytosis by PGE2 correlated with increased intracellular cyclic adenosine monophosphate (cAMP) production, and forskolin, an adenosyl cyclase agonist, confirmed the inhibitory effect of cAMP stimulation on neutrophil phagocytosis. The expression of EP2 receptors by HL-60 cells was confirmed by western blot analysis, and selective agonism of EP2 receptors mimicked the inhibition of phagocytosis by PGE2. The EP2 receptor antagonist AH-6089 partially blocked the inhibition of neutrophil phagocytosis PGE2. Specific inhibition of phosphatase and tensin homolog (PTEN) enzyme attenuated the inhibition of neutrophil phagocytosis by PGE2, and both PGE2 and increased intracellular cAMP increased neutrophil PTEN activity, which was associated with decreased PTEN phosphorylation. The results support negative regulation of the antimicrobial activity of neutrophils (i.e., phagocytosis), which has important implications for the future management of bacterial infections.

Discovery of Non-Nucleotide Small-Molecule STING Agonists via Chemotype Hybridization.

The identification of agonists of the stimulator of interferon genes (STING) pathway has been an area of intense research due to their potential to enhance innate immune response and tumor immunogenicity in the context of immuno-oncology therapy. Initial efforts to identify STING agonists focused on the modification of 2',3'-cGAMP (1) (an endogenous STING activator ligand) and other closely related cyclic dinucleotides (CDNs). While these efforts have successfully identified novel CDNs that have progressed into the clinic, their utility is currently limited to patients with solid tumors that STING agonists can be delivered to intratumorally. Herein, we report the discovery of a unique class of non-nucleotide small-molecule STING agonists that demonstrate antitumor activity when dosed intratumorally in a syngeneic mouse model.

The modulation of cAMP/PKA pathway by asiaticoside ameliorates high glucose-induced inflammation and apoptosis of retinal pigment epithelial cells.

Asiaticoside, the major bioactive constituent purified from Centella asiatica, is a pentacyclic triterpene saponin with sugar moieties (glucose-glucose-rhamnose). Its biological activities including anti-inflammation and antioxidant have been widely reported. This study aimed to investigate the role of asiaticoside in diabetic retinopathy (DR). Human retinal pigment epithelium (RPE) cells ARPE-19 were induced by high glucose. Then, cell survival rate, expression of inflammatory factors, oxidative stress, and apoptosis were measured by MTT method, western blot, oxidative stress detection kits and TUNEL respectively. To uncover the underlying mechanism, the levels of cyclic AMP (cAMP) and protein kinase A (PKA) were measured by Enzyme linked immunosorbent assay (ELISA) and PKA activities were detected by the Kemptide phosphorylation assay. Furthermore, cAMP inhibitor SQ22536 was also used to validate the mechanism. Asiaticoside suppressed the inflammation and apoptosis of ARPE-19 cells, and the activities of cAMP and PKA were inhibited upon HG induction while again released after further administration of asiaticoside. However, these effects were all abolished by SQ22536. In conclusion, we have demonstrated in this paper that asiaticoside ameliorates high glucose-induced inflammation and apoptosis of RPE cells by activating cAMP/PKA signaling pathway. asiaticoside-mediated activation of cAMP/PKA signaling pathway may serve as a potential target for the management of DR.

Single-cell analysis of prostaglandin E2-induced human decidual cell in vitro differentiation: a minimal ancestral deciduogenic signal†.

The decidua is a hallmark of reproduction in many placental mammals. Differentiation of decidual stromal cells is known to be induced by progesterone and the cyclic AMP/protein kinase A (cAMP/PKA) pathway. Several candidates have been identified as the physiological stimulus for adenylyl cyclase activation, but their relative importance remains unclear. To bypass this uncertainty, the standard approach for in vitro experiments uses membrane-permeable cAMP and progestin. We phylogenetically infer that prostaglandin E2 (PGE2) likely was the signal that ancestrally induced decidualization in conjunction with progesterone. This suggests that PGE2 and progestin should be able to activate the core gene regulatory network of decidual cells. To test this prediction, we performed a genome-wide study of gene expression in human endometrial fibroblasts decidualized with PGE2 and progestin. Comparison to a cAMP-based protocol revealed shared activation of core decidual genes and decreased induction of senescence-associated genes. Single-cell transcriptomics of PGE2-mediated decidualization revealed a distinct, early-activated state transitioning to a differentiated decidual state. PGE2-mediated decidualization was found to depend upon progestin-dependent induction of PGE2 receptor 2 (PTGER2) which in turn leads to PKA activation upon PGE2 stimulation. Progesterone-dependent induction of PTGER2 is absent in opossum, an outgroup taxon of placental mammals which is incapable of decidualization. Together, these findings suggest that the origin of decidualization involved the evolution of progesterone-dependent activation of the PGE2/PTGER2/PKA axis, facilitating entry into a PKA-dominant rather than AKT-dominant cellular state. We propose the use of PGE2 for in vitro decidualization as an alternative to 8-Br-cAMP.

Epac activation ameliorates tubulointerstitial inflammation in diabetic nephropathy.

Tubulointerstitial inflammation plays an important role in the progression of diabetic nephropathy (DN), and tubular epithelial cells (TECs) are crucial promoters of the inflammatory cascade. Exchange protein activated by cAMP (Epac) has been shown to suppress the angiotensin II (Ang-II)-induced release of inflammatory cytokines in tubular cells. However, the role of Epac in TEC-mediated tubulointerstitial inflammation in DN remains unknown. We found that administering the Epac agonist 8-pCPT-2'-O-Me-cAMP (8-O-cAMP) to db/db mice inhibited tubulointerstitial inflammation characterized by macrophage infiltration and increased inflammatory cytokine release and consequently alleviated tubulointerstitial fibrosis in the kidney. Furthermore, 8-O-cAMP administration restored CCAAT/enhancer binding protein ß (C/EBP-ß) expression and further upregulated the expression of Suppressor of cytokine signaling 3 (SOCS3), while inhibiting p-STAT3, MCP-1, IL-6, and TNF-α expression in the kidney cortex in db/db mice. And in vitro study showed that macrophage migration and MCP-1 expression induced by high glucose (HG, 30 mM) were notably reduced by 8-O-cAMP in human renal proximal tubule epithelial (HK-2) cells. In addition, 8-O-cAMP treatment restored C/EBP-ß expression in HK-2 cells and promoted C/EBP-ß translocation to the nucleus, where it transcriptionally upregulated SOCS3 expression, subsequently inhibiting STAT3 phosphorylation. Under HG conditions, siRNA-mediated knockdown of C/EBP-ß or SOCS3 in HK-2 cells partially blocked the inhibitory effect of Epac activation on the release of MCP-1. In contrast, SOCS3 overexpression inhibited HG-induced activation of STAT3 and MCP-1 expression in HK-2 cells. These findings indicate that Epac activation via 8-O-cAMP ameliorates tubulointerstitial inflammation in DN through the C/EBP-ß/SOCS3/STAT3 pathway.

cAMP controls a trafficking mechanism that maintains the neuron specificity and subcellular placement of electrical synapses.

Electrical synapses are established between specific neurons and within distinct subcellular compartments, but the mechanisms that direct gap junction assembly in the nervous system are largely unknown. Here, we show that a developmental program tunes cAMP signaling to direct the neuron-specific assembly and placement of electrical synapses in the C. elegans motor circuit. We use live-cell imaging to visualize electrical synapses in vivo and an optogenetic assay to confirm that they are functional. In ventral A class (VA) motor neurons, the UNC-4 transcription factor blocks expression of cAMP antagonists that promote gap junction miswiring. In unc-4 mutants, VA electrical synapses are established with an alternative synaptic partner and are repositioned from the VA axon to soma. cAMP counters these effects by driving gap junction trafficking into the VA axon for electrical synapse assembly. Thus, our experiments establish that cAMP regulates gap junction trafficking for the biogenesis of functional electrical synapses.

Dynamic Heterogeneity Shapes Patterns of Spiral Ganglion Activity.

Neural response properties that typify primary sensory afferents are critical to fully appreciate because they establish and, ultimately represent, the fundamental coding design used for higher-level processing. Studies illuminating the center-surround receptive fields of retinal ganglion cells, for example, were ground-breaking because they determined the foundation of visual form detection. For the auditory system, a basic organizing principle of the spiral ganglion afferents is their extensive electrophysiological heterogeneity establishing diverse intrinsic firing properties in neurons throughout the spiral ganglion. Moreover, these neurons display an impressively large array of neurotransmitter receptor types that are responsive to efferent feedback. Thus, electrophysiological diversity and its neuromodulation are a fundamental encoding mechanism contributed by the primary afferents in the auditory system. To place these features into context, we evaluated the effects of hyperpolarization and cAMP on threshold level as indicators of overall afferent responsiveness in CBA/CaJ mice of either sex. Hyperpolarization modified threshold gradients such that distinct voltage protocols could shift the relationship between sensitivity and stimulus input to reshape resolution. This resulted in an "accordion effect" that appeared to stretch, compress, or maintain responsivity across the gradient of afferent thresholds. cAMP targeted threshold and kinetic shifts to rapidly adapting neurons, thus revealing multiple cochleotopic properties that could potentially be independently regulated. These examples of dynamic heterogeneity in primary auditory afferents not only have the capacity to shift the range, sensitivity, and resolution, but to do so in a coordinated manner that appears to orchestrate changes with a seemingly unlimited repertoire.SIGNIFICANCE STATEMENT How do we discriminate the more nuanced qualities of the sound around us? Beyond the basics of pitch and loudness, aspects, such as pattern, distance, velocity, and location, are all attributes that must be used to encode acoustic sensations effectively. While higher-level processing is required for perception, it would not be unexpected if the primary auditory afferents optimized receptor input to expedite neural encoding. The findings reported herein are consistent with this design. Neuromodulation compressed, expanded, shifted, or realigned intrinsic electrophysiological heterogeneity to alter neuronal responses selectively and dynamically. This suggests that diverse spiral ganglion phenotypes provide a rich substrate to support an almost limitless array of coding strategies within the first neural element of the auditory pathway.

Mitogen-induced transcriptional programming in human fibroblasts.

Treatment of serum-starved quiescent human cells with fetal bovine serum (FBS), epidermal growth factor (EGF), or the phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA) activates the RAS-MAPK pathway which initiates a transcriptional program which drives cells toward proliferation. Stimulation of the RAS-MAPK pathway activates mitogen- and stress-activated kinases (MSK) 1 and 2, which phosphorylate histone H3 at S10 (H3S10ph) or S28 (H3S28ph) (nucleosomal response) located at the regulatory regions of immediate-early genes, setting in motion a series of chromatin remodeling events that result in transcription initiation. To investigate immediate-early genes regulated by the MSK, we have completed transcriptome analyses (RNA sequencing) of human normal fibroblast cells (CCD-1070Sk) stimulated with EGF or TPA ± H89, a potent MSK/PKA inhibitor. The induction of many immediate-early genes was independent of MSK activity. However, the induction of immediate-early genes attenuated with H89 also had reduced induction with the PKA inhibitor, Rp-cAMPS. Several EGF-induced genes, coding for transcriptional repressors, were further upregulated with H89 but not with Rp-cAMPS, suggesting a role for MSK in modulating the induction level of these genes.