Decrease In cAMP Levels Research Articles

NPFF Decreases Activity of Human Arcuate NPY Neurons: A Study in Embryonic-Stem-Cell-Derived Model.

Restoring the control of food intake is the key to obesity management and prevention. The arcuate nucleus (ARC) of the hypothalamus is extensively being studied as a potential anti-obesity target. Animal studies showed that neuropeptide FF (NPFF) reduces food intake by its action in neuropeptide Y (NPY) neurons of the hypothalamic ARC, but the detailed mode of action observed in human neurons is missing, due to the lack of a human-neuron-based model for pharmacology testing. Here, we validated and utilized a human-neural-stem-cell-based (hNSC) model of ARC to test the effects of NPFF on cellular pathways and neuronal activity. We found that in the human neurons, decreased cAMP levels by NPFF resulted in a reduced rate of cytoplasmic calcium oscillations, indicating an inhibition of ARC NPY neurons. This suggests the therapeutic potential of NPFFR2 in obesity. In addition, we demonstrate the use of human-stem-cell-derived neurons in pharmacological applications and the potential of this model to address functional aspects of human hypothalamic neurons.

Exploring the role of cAMP in gabapentin- mediated pain attenuating effects in chronic constriction injury model in rats

It has been shown that an increase in cAMP leads to pain sensitization and gabapentin is shown to decrease cAMP levels. However, the impact of drugs modulating cAMP levels on analgesic actions of gabapentin is not studied. The present study investigates the effect of milrinone on pain attenuating effects of gabapentin in chronic constriction injury (CCI). Neuropathic pain was induced by putting four loose ligatures around the sciatic nerve. The pain assessment was done by noting the paw withdrawal threshold in the pinprick test, paw withdrawal latency in hot plate test and paw withdrawal duration in acetone drop test before surgery and on 14th- day post-surgery. There was a significant development of cold allodynia, mechanical and heat hyperalgesia on 14th day in CCI rats. Gabapentin (100 mg/kg) treatment for 14 days significantly attenuated pain, while milrinone (50 mg/kg) treatment for 14 days significantly exacerbated neuropathic pain in CCI-subjected rats. Milrinone (30 and 50 mg/kg) also attenuated analgesic actions of gabapentin in CCI-subjected rats, suggests that gabapentin may abolish neuropathic pain by increasing the intracellular levels of cAMP in CCI-subjected rats.

Upregulation of Phosphodiesterase 2A Augments T Cell Activation by Changing cGMP/cAMP Cross-Talk.

3′,5′-cyclic adenosine monophosphate (cAMP) is well-known for its diverse immunomodulatory properties, primarily inhibitory effects during T cell activation, proliferation, and production of pro-inflammatory cytokines. A decrease in cAMP levels, due to the hydrolyzing activity of phosphodiesterases (PDE), is favoring inflammatory responses. This can be prevented by selective PDE inhibitors, which makes PDEs important therapeutic targets for autoimmune disorders. In this study, we investigated the specific roles of PDE2A and PDE3B in the regulation of intracellular cAMP levels in different mouse T cell subsets. Unexpectedly, T cell receptor (TCR) activation led to a selective upregulation of PDE2A at the protein level in conventional T cells (Tcon), whereas no changes were detected in regulatory T cells (Treg). In contrast, protein expression of PDE3B was significantly higher in both non-activated and activated Tcon subsets as compared to Treg, with no changes upon TCR engagement. Live-cell imaging of T cells expressing a highly sensitive Förster resonance energy transfer (FRET)-based biosensor, Epac1-camps, has enabled cAMP measurements in real time and revealed stronger responses to the PDE2A inhibitors in activated vs non-activated Tcon. Importantly, stimulation of intracellular cGMP levels with natriuretic peptides led to an increase of cAMP in non-activated and a decrease of cAMP in activated Tcon, suggesting that TCR activation changes the PDE3B-dependent positive to PDE2A-dependent negative cGMP/cAMP cross-talk. Functionally, this switch induced higher expression of early activation markers CD25 and CD69. This constitutes a potentially interesting feed-forward mechanism during autoimmune and inflammatory responses that may be exploited therapeutically.

Phosphatidic acid-PKA signaling regulates p38 and ERK1/2 functions in ligand-independent EGFR endocytosis.

Ligand-independent epidermal growth factor receptor (EGFR) endocytosis is inducible by a variety of stress conditions converging upon p38 kinase. A less known pathway involves phosphatidic acid (PA) signaling toward the activation of type 4 phosphodiesterases (PDE4) that decrease cAMP levels and protein kinase A (PKA) activity. This PA/PDE4/PKA pathway is triggered with propranolol used to inhibit PA hydrolysis and induces clathrin-dependent and clathrin-independent endocytosis, followed by reversible accumulation of EGFR in recycling endosomes. Here we give further evidence of this signaling pathway using biosensors of PA, cAMP, and PKA in live cells and then show that it activates p38 and ERK1/2 downstream the PKA inhibition. Clathrin-silencing and IN/SUR experiments involved the activity of p38 in the clathrin-dependent route, while ERK1/2 mediates clathrin-independent EGFR endocytosis. The PA/PDE4/PKA pathway selectively increases the EGFR endocytic rate without affecting LDLR and TfR constitute endocytosis. This selectiveness is probably because of EGFR phosphorylation, as detected in Th1046/1047 and Ser669 residues. The EGFR accumulates at perinuclear recycling endosomes colocalizing with TfR, fluorescent transferrin, and Rab11, while a small proportion distributes to Alix-endosomes. A non-selective recycling arrest includes LDLR and TfR in a reversible manner. The PA/PDE4/PKA pathway involving both p38 and ERK1/2 expands the possibilities of EGFR transmodulation and interference in cancer.

β3 adrenergic receptor as potential therapeutic target in ADPKD.

Autosomal dominant polycystic kidney disease (ADPKD) disrupts renal parenchyma through progressive expansion of fluid‐filled cysts. The only approved pharmacotherapy for ADKPD involves the blockade of the vasopressin type 2 receptor (V2R). V2R is a GPCR expressed by a subset of renal tubular cells and whose activation stimulates cyclic AMP (cAMP) accumulation, which is a major driver of cyst growth. The β3‐adrenergic receptor (β3‐AR) is a GPCR expressed in most segments of the murine nephron, where it modulates cAMP production. Since sympathetic nerve activity, which leads to activation of the β3‐AR, is elevated in patients affected by ADPKD, we hypothesize that β3‐AR might constitute a novel therapeutic target. We find that administration of the selective β3‐AR antagonist SR59230A to an ADPKD mouse model (Pkd1fl/fl;Pax8rtTA;TetO‐Cre) decreases cAMP levels, producing a significant reduction in kidney/body weight ratio and a partial improvement in kidney function. Furthermore, cystic mice show significantly higher β3‐AR levels than healthy controls, suggesting a correlation between receptor expression and disease development. Finally, β3‐AR is expressed in human renal tissue and localizes to cyst‐lining epithelial cells in patients. Thus, β3‐AR is a potentially interesting target for the development of new treatments for ADPKD.

Dermal Olfactory Receptor OR51B5 Is Essential for Survival and Collagen Synthesis in Human Dermal Fibroblast (Hs68 Cells).

Skin dermis comprises extracellular matrix components, mainly collagen fibers. A decrease in collagen synthesis caused by several factors, including ultraviolet (UV) irradiation and stress, eventually causes extrinsic skin aging. Olfactory receptors (ORs) were initially considered to be specifically expressed in nasal tissue, but several ORs have been reported to be present in other tissues, and their biological roles have recently received increasing attention. In this study, we aimed to characterize the role of ORs in cell survival and collagen synthesis in dermal fibroblasts. We confirmed that UVB irradiation and dexamethasone exposure significantly decreased cell survival and collagen synthesis in Hs68 dermal fibroblasts. Moreover, we demonstrated that the mRNA expression of 10 ORs detectable in Hs68 cells was significantly downregulated in aged conditions compared with that in normal conditions. Thereafter, by individual knockdown of the 10 candidate ORs, we identified that only OR51B5 knockdown leads to a reduction of cell survival and collagen synthesis. OR51B5 knockdown decreased cAMP levels and dampened the downstream protein kinase A/cAMP-response element binding protein pathway, downregulating the survival- and collagen synthesis-related genes in the dermal fibroblasts. Therefore, OR51B5 may be an interesting candidate that plays a role in cell survival and collagen synthesis.

Nitration of protein kinase G-Iα modulates cyclic nucleotide crosstalk via phosphodiesterase 3A: Implications for acute lung injury

Phosphodiesterase 3A (PDE3A) selectively cleaves the phosphodiester bond of cAMP and is inhibited by cGMP, making it an important regulator of cAMP–cGMP signaling crosstalk in the pulmonary vasculature. In addition, the nitric oxide–cGMP axis is known to play an important role in maintaining endothelial barrier function. However, the potential role of protein kinase G-Iα (PKG-Iα) in this protective process is unresolved and was the focus of our study. We describe here a novel mechanism regulating PDE3A activity, which involves a PKG-Iα–dependent inhibitory phosphorylation of PDE3A at serine 654. We also show that this phosphorylation is critical for maintaining intracellular cAMP levels in the pulmonary endothelium and endothelial barrier integrity. In an animal model of acute lung injury (ALI) induced by challenging mice with lipopolysaccharide (LPS), an increase in PDE3 activity and a decrease in cAMP levels in lung tissue was associated with reduced PKG activity upon PKG-Iα nitration at tyrosine 247. The peroxynitrite scavenger manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin prevented this increase in PDE3 activity in LPS-exposed lungs. In addition, site-directed mutagenesis of PDE3A to replace serine 654 with alanine yielded a mutant protein that was insensitive to PKG-dependent regulation. Taken together, our data demonstrate a novel functional link between nitrosative stress induced by LPS during ALI and the downregulation of barrier-protective intracellular cAMP levels. Our data also provide new evidence that PKG-Iα is critical for endothelial barrier maintenance and that preservation of its catalytic activity may be efficacious in ALI therapy.

P2Y 14 receptor regulates adipocyte lipid mobilization and whole‐body glucose homeostasis

The incidence and prevalence of obesity and type 2 diabetes (T2D) is rapidly increasing worldwide. Obesity is associated with chronically elevated plasma free fatty acid (FFA) levels, causing peripheral insulin resistance and T2D. Drugs that are able to regulate adipocyte lipolytic processes, improving insulin sensitivity and whole-body glucose homeostasis, have considerable therapeutic potential. Gi-coupled P2Y14 receptor (P2Y14R), activated by UDP-glucose is abundantly expressed in mouse and human adipocytes. P2Y14R activation in primary mouse adipocytes decreased cAMP levels and CL-316,243 (β3-adrenoceptor agonist) induced lipolysis. To investigate the physiological relevance of adipocyte P2Y14Rs in regulating lipid and glucose homeostasis in-vivo, we generated a mouse model lacking P2Y14R specifically in adipocytes. Lack of adipocyte P2Y14R increased lipolysis only in the fasting state, resulting in decreased body weight, improved glucose tolerance and insulin sensitivity. Accordingly, high fat diet increased, whereas fasting decreased P2Y14R expression in visceral white adipocytes. Mechanistic studies suggested that activation of P2Y14R reduced the activity of lipolytic enzymes including ATGL and HSL, thereby inhibiting lipolysis. Supporting these findings, administration of P2Y14R agonist in control mice decreased lipolysis. Coadministration of P2Y14R antagonist with the P2Y14R agonist rescued the decrease in lipolysis. In conclusion, we demonstrate that lack of adipocyte P2Y14R decreased diet-induced obesity by enhancing lipolysis in the fasting state, suggesting that P2Y14R antagonists may prove beneficial for the therapy of obesity and T2D.

Plasmodium falciparum sexual parasites regulate infected erythrocyte permeability

To ensure the transport of nutrients necessary for their survival, Plasmodium falciparum parasites increase erythrocyte permeability to diverse solutes. These new permeation pathways (NPPs) have been extensively characterized in the pathogenic asexual parasite stages, however the existence of NPPs has never been investigated in gametocytes, the sexual stages responsible for transmission to mosquitoes. Here, we show that NPPs are still active in erythrocytes infected with immature gametocytes and that this activity declines along gametocyte maturation. Our results indicate that NPPs are regulated by cyclic AMP (cAMP) signaling cascade, and that the decrease in cAMP levels in mature stages results in a slowdown of NPP activity. We also show that NPPs facilitate the uptake of artemisinin derivatives and that phosphodiesterase (PDE) inhibitors can reactivate NPPs and increase drug uptake in mature gametocytes. These processes are predicted to play a key role in P. falciparum gametocyte biology and susceptibility to antimalarials.

Abstract PR17: Metabolic re-wiring in African-American prostate cancer: A role of adenosine-inosine axis

Abstract African-American (AA) men have a 60% higher incidence of prostate cancer compared to European-American (EA) men and tend to have a more aggressive clinical outcome. An understanding of the altered biological responses and the factors that lead to health disparity in prostate cancer development and progression is unclear. Metabolism defines the physiological state of the cell and metabolic reprogramming is a hallmark of cancer. Thus, studying the metabolic landscape will be critical for understanding the biological changes that might contribute to this disparity. Analysis of 190 metabolites across prostate cancer/benign adjacent tissue pairs from ancestry typed AA and EA men performed in our laboratory, revealed altered levels of nucleosides adenosine and inosine. High inosine to adenosine ratio is observed in AA men compared to EA men. In line with this finding, the enzyme adenosine deaminase (ADA), which converts adenosine to inosine was found elevated in AA men, potentially explaining the high inosine to adenosine ratio in AA PCa. The consequences of the accumulation of these metabolites on AA PCa progression are unknown. The current study will address the knowledge gap on the consequences of elevated ADA activity in AA PCa and attempt to dissect its role in effecting an aggressive phenotype in PCa. To determine the role of elevated ADA in PCa, it was overexpressed in ancestry-typed AA (MDA-PCa-2A) and EA PCa(LNCaP) cell lines (termed ADA OE). Phenotypic examination of these cells revealed increased ADA enzyme activity decreases the cell-ECM adhesion. This adhesion decrease is facilitated by a decrease in cell adhesion protein integrin β1 (ITGB1). Elevated ADA enzyme activity decreases cAMP which mediates this adhesion decrease by disrupting the Epac-Rap1 pathway. It is postulated that high inosine upon ADA OE activates adenosine receptors A1 and A3, which causes this decrease in cAMP levels. These molecular findings suggest that ADA-mediated adhesion decrease could facilitate metastatic dissemination from the primary tumor. From a translational viewpoint, ADA enzyme activity could serve as a biomarker for metastatic prostate cancer. Therefore, we further look to analyze the inosine to adenosine levels (ADA enzyme activity) in patient samples and correlate with various clinical outcomes in PCa. Our initial analysis reveals high inosine to adenosine ratio in biopsy positive patient samples compared to biopsy negative samples. Our next steps of analysis include correlating the enzyme activity with (i) Biochemical Recurrence (ii) Time to attaining castration resistance in castration sensitive patients (iii) Time to metastasis (iv) Response to Androgen deprivation in patients after BCr. Based on our current findings, we expect inosine to adenosine ratio in plasma or urine to serve as a predictive marker for PCa incidence or progression in AA men. In addition, we expect ADA to serve as a potential therapeutic target for AA PCa. Citation Format: Christy Charles, Jie Gohlke, Stacy Lloyd, Uttam Rasaily, James Henderson, Balasubramanyam Karanam, Brian Simons, Nora Navone, Rick Kittles, Stefan Ambs, George Michailidis, Nagireddy Putluri, Arun Sreekumar. Metabolic re-wiring in African-American prostate cancer: A role of adenosine-inosine axis [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PR17.

Di (2-ethylhexyl) phthalate impairs primordial follicle assembly by increasing PDE3A expression in oocytes.

It is known that Di (2-ethylhexyl) phthalate (DEHP) may impact mammalian reproduction and that in females one target of the drug's action is follicle assembly. Here we revisited the phthalate's action on the ovary and from bioinformatics analyses of the transcriptome performed on newborn mouse ovaries exposed invitro to DEHP, up-regulation of PDE3A, as one of the most important alterations caused by DEHP on early folliculogenesis, was identified. We obtained some evidence suggesting that the decrease of cAMP level in oocytes and the parallel decrease of PKA expression, consequent on the PDE3A increase, were a major cause of the reduction of follicle assembly in the DEHP-exposed ovaries. In fact, Pde3a RNAi on cultured ovaries reducing cAMP and PKA decrease counteracted the primordial follicle assembly impairment caused by the compound. Moreover, RNAi normalized the level of Kit, Nobox, Figla mRNA and GDF9, BMP15, CX37, γH2AX proteins in oocytes, and KitL transcripts in granulosa cells as well as their proliferation rate altered by DEHP exposure. Taken together, these results identify PDE3A as a new critical target of the deleterious effects of DEHP on early oogenesis in mammals and highlight cAMP-dependent pathways as major regulators of oocyte and granulosa cell activities crucial for follicle assembly. Moreover, we suggest that the level of intracellular cAMP in the oocytes may be an important determinant for their capability to repair DNA lesions caused by DNA damaging compounds including DEHP.

Dexamethasone rescues TGF-\u03b21-mediated \u03b22-adrenergic receptor dysfunction and attenuates phosphodiesterase 4D expression in human airway smooth muscle cells

Glucocorticoids (GCs) and β2-adrenergic receptor (β2AR) agonists improve asthma outcomes in most patients. GCs also modulate gene expression in human airway smooth muscle (HASM), thereby attenuating airway inflammation and airway hyperresponsiveness that define asthma. Our previous studies showed that the pro-fibrotic cytokine, transforming growth factor- β1 (TGF-β1) increases phosphodiesterase 4D (PDE4D) expression that attenuates agonist-induced levels of intracellular cAMP. Decreased cAMP levels then diminishes β2 agonist-induced airway relaxation. In the current study, we investigated whether glucocorticoids reverse TGF-β1-effects on β2-agonist-induced bronchodilation and modulate pde4d gene expression in HASM. Dexamethasone (DEX) reversed TGF-β1 effects on cAMP levels induced by isoproterenol (ISO). TGF-β1 also attenuated G protein-dependent responses to cholera toxin (CTX), a Gαs stimulator downstream from the β2AR receptor. Previously, we demonstrated that TGF-β1 treatment increased β2AR phosphorylation to induce hyporesponsiveness to a β2 agonist. Our current data shows that expression of grk2/3, kinases associated with attenuation of β2AR function, are not altered with TGF-β1 stimulation. Interestingly, DEX also attenuated TGF-β1-induced pde4d gene expression. These data suggest that steroids may be an effective therapy for treatment of asthma patients whose disease is primarily driven by elevated TGF-β1 levels.

Abstract 5027: The mu-opioid receptor antagonist methylnaltrexone can decrease tumorigenesis in head and neck squamous cell carcinoma

Abstract Head and neck cancer (HNC) is the sixth leading cause of cancer in the world, and the third most common neoplasia in developing countries [1]. Patients with HNC may suffer significant pain, for which opioids are commonly given. However, recent reports suggest that opioids may promote cancer progression by action on the mu-opioid receptor (MOR) [2, 3]. The goal of this research was to test the hypothesis that the MOR antagonist methylnaltrexone (MTNX) can decrease tumorigenesis in HN squamous cell carcinoma (SCC). First, we confirmed via RNAseq and western blot analyses that FaDu cells (a HNSCC line) express MOR. Then, we performed cyclic adenosine monophosphate (cAMP) activation enzyme-linked immunosorbent assay to evaluate the activity of MOR after activation with [D-Ala2N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO), a selective MOR agonist. Short term (1hr) treatment with DAMGO decreased cAMP levels, suggesting full activity of the receptor. We conducted similar cAMP activity assays using MNTX. Our dose-dependent (10nM-1µM) experiments revealed that 1hr exposure of cells to MTNX inhibited cAMP activity, but not after 48 hrs of treatment, consistent with an inverse or partial agonist effect at low doses. To assess the impact of our findings on FaDu cell behaviors, we conducted in vitro proliferation, colony formation, and migration assays. MTNX at different concentrations (0.1nM, 1nM, 10nM, 100nM and 1µM) inhibited FaDu cell proliferative and metastatic cell behaviors (p<0.05). To translate our results to in vivo, we inoculated the tongue of athymic nude mice with FaDu cells (200,000 cells/30 mL). Starting the day of cell inoculation, we randomly treated the animals with MNTX (subcutaneously, 1µg/kg, 10 µg/kg, and 100µg/kg) versus placebo (saline only) for 17 consecutive days. In vivo experiments demonstrated that administration of MTNX at three different concentrations delayed tumor progression and reduced tumor growth and formation compared to placebo (p<0.0001). Overall, our results suggest that MTNX inhibits tumorigenesis of HNSCC, possibly by acting on MOR. 1. Siegel, R.L. et al, Cancer statistics, 2018. CA Cancer J Clin, 2018. 68(1): p. 7-30. 2. Lennon, F.E., et al, The mu-opioid receptor in cancer progression: is there a direct effect? Anesthesiology, 2012. 116(4): p. 940-5. 3. Patino, M.A., et al., The impact of intraoperative opioid use on survival after oral cancer surgery. Oral Oncol, 2017. 74: p. 1-7. Citation Format: Aysegul Gorur, Miguel Patino, Ted Shi, Hideaki Takahashi, Jeffrey N. Myers, Juan P. Cata. The mu-opioid receptor antagonist methylnaltrexone can decrease tumorigenesis in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5027.

Abstract IA26: Oncogenic LncRNA promotes escape from intrinsic Hippo tumor suppression

Abstract How tumor cells genetically lose antigenicity and evade intrinsic tumor suppression barriers has been uncertain for decades. Here, we report that tissue-specific expression of a human long noncoding RNA LINK-A in mouse mammary gland initiates metastatic breast tumor, which models human basal-like breast cancer morphologically, genetically, transcriptionally, and metabolically. LINK-A expression facilitates crosstalk between PIP3 and inhibitory GPCRs pathways, leading to decreased cAMP level and protein kinase A (PKA)-mediated phosphorylation of E3 ubiquitin ligase TRIM71. This leads to enhanced K48-polyubiquitination-mediated degradation of tumor suppressor MST1 as well as LATS1. Treatment with LINK-A antisense oligonucleotides or Rauwolscine (a Galphai antagonist) stabilizes Hippo tumor suppressor pathway, sensitizing breast tumors to immune checkpoint blockers. PD-1 blockade-resistant TNBC patients exhibited elevated LINK-A and downregulated Hippo tumor suppressors. We demonstrate an lncRNA-dependent escape from intrinsic tumor suppression and immunosurveillance, which provides a regimen of combinational immunotherapy and effective early prevention for TNBC. Citation Format: Qingsong Hu, Chunru Lin, Liuqing Yang. Oncogenic LncRNA promotes escape from intrinsic Hippo tumor suppression [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr IA26.

Abstract A095: Metabolic rewiring in African-American prostate cancer: A role for adenosine-inosine axis in tumor progression

Abstract Introduction: Prostate cancer (PCa) prevalence and mortality are remarkably higher in African-American (AA)men compared to European-American (EA) men. In addition to known differences in socioeconomic status and access to health care, there is increasing evidence to suggest biological differences between AA and EA PCa. Our lab has pioneered the understanding of reprogrammed metabolism, a hallmark of tumor progression, in AA and EA PCa. Analysis of a compendium of 190 metabolites in ancestry verified, pathologically confirmed AA and EA PCa tissues followed by validation of key pathway components in plasma and urine revealed high inosine to adenosine ratio in AA PCa compared to EA PCa. Consistent with this, the enzyme adenosine deaminase (ADA), which converts adenosine to inosine was also found to be elevated in AA PCa. The current study will address the role of adenosine-inosine axis in AA PCa progression and attempt to delineate its significance in the clinical setting. Methods: Analysis of metabolites in tissues, plasma and urine was done using LC/MS. Tissue microarray analysis (TMA) was done to check the expression of ADA in tissues and correlate it to clinical outcomes. To determine the function of ADA in PCa progression, the gene was overexpressed in MDA-PCa-2A (AA) and LNCaP (EA) cells using lentiviral transduction (termed ADA OE). In vitro invasion was assessed using Boyden chamber assay and in vivo tumorigenic properties were examined in nude mice. Molecular studies were performed using qPCR, ELISA and western blotting. Results: Overexpression of the enzyme ADA in PCa cells confers an anchorage-independent growth phenotype (a.k.a. Anoikis Resistance) associated with a reduction in Integrin β1 (ITGB1) levels. Anoikis resistance is a key prerequisite for metastasis and a hallmark of circulating tumor cells (CTCs). ADA OE AA cells exhibited higher invasive potential compared to the EA cells. Molecular analysis revealed downregulation E-cadherin (epithelial marker) and Bim (pro-apoptotic marker), and upregulation of XIAP (anti-apoptotic marker), all of which support anoikis resistance phenotype in these cells. ADA OE cells had reduced cAMP levels, and addition of external cAMP resulted in reversal of anoikis resistance, increased cell adhesion and rescued ITGB1 levels. Mechanistically, it is postulated that high inosine upon ADA OE activates adenosine receptors A1 and A3, which causes a decrease in cAMP levels. Decreased cAMP affects the Epac-Rap1 signaling, a key regulator of integrin-mediated adhesion. Concomitantly, ADA OE cells also showed a decrease in Rap1 levels. Conclusion: Elevated inosine to adenosine ratio associated with increased expression of ADA is a metabolic hallmark in AA PCa, associated with anoikis resistance and possibly increased CTCs. Our findings also strongly support the potential of using inosine to adenosine ratio in plasma or urine as a predictive marker for PCa incidence or progression in AA men. In addition, ADA could serve as a potential therapeutic target for AA PCa. Citation Format: Christy Charles, Jie Gohlke, Stacy Lloyd, James Henderson, Balasubramaniam Karanam, Nora Navone, Rick Kittles, Stefan Ambs, George Michailidis, Nagireddy Putluri, Arun Sreekumar. Metabolic rewiring in African-American prostate cancer: A role for adenosine-inosine axis in tumor progression [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr A095.

Mitochondrial stress-activated cGAS-STING pathway inhibits thermogenic program and contributes to overnutrition-induced obesity in mice

Obesity is a global epidemic that is caused by excessive energy intake or inefficient energy expenditure. Brown or beige fat dissipates energy as heat through non-shivering thermogenesis by their high density of mitochondria. However, how the mitochondrial stress-induced signal is coupled to the cellular thermogenic program remains elusive. Here, we show that mitochondrial DNA escape-induced activation of the cGAS-STING pathway negatively regulates thermogenesis in fat-specific DsbA-L knockout mice, a model of adipose tissue mitochondrial stress. Conversely, fat-specific overexpression of DsbA-L or knockout of STING protects mice against high-fat diet-induced obesity. Mechanistically, activation of the cGAS-STING pathway in adipocytes activated phosphodiesterase PDE3B/PDE4, leading to decreased cAMP levels and PKA signaling, thus reduced thermogenesis. Our study demonstrates that mitochondrial stress-activated cGAS-STING pathway functions as a sentinel signal that suppresses thermogenesis in adipose tissue. Targeting adipose cGAS-STING pathway may thus be a potential therapeutic strategy to counteract overnutrition-induced obesity and its associated metabolic diseases.

Striatal specific orphan GPCRs crosstalk with Dopamine D2 receptors to regulate cAMP signaling

The striatum is a subcortical brain region that performs vital functions including movement control, regulation of attention and is a critical component of the reward system. Current drug targets have displayed limited efficacy in treating striatal‐related diseases including for substance‐use disorders, schizophrenia and Parkinson’s disease. G protein‐coupled receptors (GPCRs) are valuable drug targets with ~35% of FDA approved medicines targeting these receptors. However, nearly 120 GPCRs remain orphan receptors, whose endogenous ligands, G protein signaling, and therapeutic potential are unknown. We previously conducted a comprehensive RNAseq genome‐wide survey of human tissue gene expression and identified several orphan GPCRs selectively expressed in the human striatum, namely, GPR6, 52, 88, 101, 139 and 149. Here we elucidate striatal orphan receptors that modulate G protein‐mediated cAMP signaling and examine potential signaling crosstalk between orphan receptors and dopamine D2 receptors (D2R). Biochemical and CRISPR/Cas9 knockout studies in human embryonic kidney 293 (HEK293) cells confirmed GPR6, 52 and 101 increased cyclic adenosine monophosphate (cAMP) via Gs/olf G proteins, while GPR88 decreased cAMP levels via Gi/o G proteins. Notably, when expressed alone, GPR6, 52 and 101 profoundly increased basal levels of cAMP, indicating these receptors are constitutively active. We hypothesized that GPR6, 52, and 101 may set the basal tone for cAMP that would allow robust function of the Gi/o‐coupled dopamine D2R to inhibit adenylyl cyclase and decrease cAMP. When expressed alone in HEK293 cells and without any stimulation to the adenylyl cyclase system, the cAMP inhibition window for the D2R and agonist quinpirole was minimal and difficult to reliably detect (~1000 light counts/sec, Glosensor assay). However, when the D2R was co‐expressed with either GPR6, 52, or 101, the assay window to inhibit cAMP for quinpirole was magnified by over 200X (200,000–500,000 light counts/sec), with agonist potency unchanged. These studies suggest that these striatal specific human orphan receptors may establish dynamic basal cAMP levels and this may facilitate inhibition of cAMP via Gi/o‐coupled receptors, such as the D2R. Our findings also suggest GPR6, GPR52 and GPR101 are potential drug targets to modulate cAMP signaling in striatal specific neurons, which may be therapeutic for treating several neurological and neuropsychiatric diseases.Support or Funding InformationNIDA T32 DA07287 (DEF) and RSGPT18‐ PhRMA Foundation (JAA).

GPR64 promotes cAMP pathway in tumor aggressiveness in sparsely granulated growth hormone cell adenomas

There is an increasing agreement that acromegaly caused by growth hormone (GH) cell adenoma has two distinct subtypes: densely granulated (DG) and sparsely granulated (SG). We hypothesized that differential molecular signatures may explain their behavior. Total transcriptome sequencing was performed on ten DG and seven SG adenomas. The differentially expressed RNAs were identified by bioinformatic analyses, and a candidate RNA was verified by quantitative real-time PCR. Immunohistochemical staining was also performed to detect the protein expression of the candidate. Clinical parameters were correlated with protein expression. Subsequently, cell proliferation, colony formation, and cell cycle progression were analyzed after knockdown of the candidate in pituitary GH3 cells. Activation of the cAMP pathway was assessed by ELISA and Western blot. We confirmed that there were obvious differentially expressed genes between the subtypes. Through gene profiling, we discovered that an orphan adhesion G protein-coupled receptor, GPR64, was overexpressed in more aggressive SG adenomas. Noticeably, GPR64 knockdown significantly inhibited the proliferation of GH3 tumor cells and decreased colony formation. The knockdown also induced cell cycle arrest in GH3 tumor cells. Further studies revealed that GPR64 knockdown decreased cAMP levels and the ratios of p-CREB/CREB, indicating that it suppressed the cAMP/CREB pathway. Our results indicated that GPR64 may promote aggressiveness in SG-type GH cell adenomas and that it is a key factor regulating the cAMP pathway to promote aggressiveness of GH cell adenomas.

The meaning of non-classical estrogen receptors and peroxisome proliferator-activated receptor for boar Leydig cell of immature testis

Communication in biological systems involves diverse-types of cell-cell interaction including cross-talk between receptors expressed by the target cells. Recently, novel sort of estrogen receptors (G protein - coupled estrogen receptor; GPER and estrogen-related receptor; ERR) that signal directly via estrogen binding and/or via mutual interaction-regulated estrogen signaling were reported in various organs including testis. Peroxisome proliferator - activated receptor (PPAR) is responsible for maintaining of lipid homeostasis that is critical for sex steroid production in the testis. Here, we investigated the role of interaction between GPER, ERRβ and PPARγ in steroidogenic Leydig cells of immature boar testis. Testicular fragments cultured ex vivo were treated with GPER or PPARγ antagonists. Then, cell ultrastructure, expression and localization of GPER, ERRβ, PPARγ together with the molecular receptor mechanism, through cyclic AMP and Raf/Ras/extracellular signal activated kinases (ERK), in the control of cholesterol concentration and estrogen production by Leydig cells were studied. In the ultrastructure of antagonist-treated Leydig cells, mitochondria were not branched and not bifurcated as they were found in control. Additionally, in PPARγ-blocked Leydig cells changes in the number of lipid droplets were revealed. Independent of used antagonist, western blot revealed decreased co-expression of GPER, ERRβ, PPARγ with exception of increased expression of ERRβ after PPARγ blockage. Immunohistochemistry confirmed presence of all receptors partially located in the nucleus or cytoplasm of Leydig cells of both control and treated testes. Changes in receptor expression, decreased cholesterol and increased estradiol tissue concentrations occurred through decreased cAMP level (with exception after GPER blockage) as well as Raf/Ras/ERK pathway expression. These all findings indicate that GPER-ERRβ-PPARγ interaction exists in immature boar testis and regulates Leydig cell function. Further detailed studies and considerations on GPER-ERRβ-PPARγ as possible diagnosis/therapy target in disturbances of testis steroidogenic function are needed.

Increased miR-142-3p Expression Might Explain Reduced Regulatory T Cell Function in Granulomatosis With Polyangiitis.

Objectives: Regulatory T cells (Tregs) are frequently functionally impaired in patients with granulomatosis with polyangiitis (GPA). However, the mechanism underlying their impaired function is unknown. Here, we hypothesized that Treg dysfunction in GPA is due to altered microRNA (miRNA) expression.Methods: RNA isolated from FACS-sorted memory (M) Tregs (CD4+CD45RO+CD25+CD127−) of 8 healthy controls (HCs) and 8 GPA patients without treatment was subjected to miRNA microarray analysis. Five differentially expressed miRNAs were validated in a larger cohort by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). An miRNA target gene database search revealed targets that were tested with RT-qPCR in MTregs from patients and HCs. cAMP levels were measured using flow cytometry.Results: Microarray analysis revealed 19 differentially expressed miRNAs, of which miR-142-3p was confirmed to be significantly upregulated in MTregs from GPA patients compared to those from HCs (1.9-fold, p = 0.03). In vitro overexpression of miR-142-3p lowered the suppressive capacity of MTregs (2.1-fold, p = 0.03), and miR-142-3p expression correlated negatively with the suppressive capacity (rho = −0.446, p = 0.04). Overexpression of miR-142-3p significantly decreased cAMP levels (p = 0.02) and tended to decrease the mRNA levels of a predicted target gene, adenylate cyclase 9 (ADCY9; p = 0.06). In comparison to those from HCs, MTregs from GPA patients had lower ADCY9 mRNA levels (2-fold, p = 0.008) and produced significantly less cAMP after stimulation. Importantly, induction of cAMP production in miR-142-3p overexpressed MTregs by forskolin restored their suppressive function in vitro.Conclusion: Overexpression of miR-142-3p in MTregs from GPA patients might cause functional impairment by targeting ADCY9, which leads to the suppression of cAMP production.