PKA Inhibitor Research Articles

Apremilast-induced increases in acute ethanol intoxication and decreases in ethanol drinking in mice involve PKA phosphorylation of GABAA β3 subunits

We previously showed that apremilast, an FDA-approved PDE4 inhibitor, selectively alters behavioral responses to ethanol and certain GABAergic drugs in a PKA-dependent manner in C57BL6/J mice. Here, we investigated if PKA phosphorylation of β3 GABAA receptor subunits is involved in apremilast regulation of ethanol, propofol, or diazepam responses. Apremilast prolonged rotarod ataxia and loss of the righting reflex by ethanol and propofol in wild-type mice, but not in β3-S408A/S409A knock-in mice. In contrast, apremilast hastened recovery from the ataxic and sedative effects of diazepam in both genotypes. These findings suggest that apremilast modulation of ethanol and propofol behaviors in wild-type mice is mediated by β3 subunit phosphorylation, whereas its actions on diazepam responses involve a different mechanism. The PKA inhibitor H-89 prevented apremilast modulation of ethanol-induced ataxia. Apremilast sensitized wild-type males to ethanol-induced ataxia and decreased acute functional tolerance (AFT) in females but had no effect in β3-S408A/S409A mice of either sex. These results could not be attributed to genotype differences in blood ethanol clearance. There were also no baseline genotype differences in ethanol consumption and preference in two different voluntary drinking procedures. However, the ability of apremilast to reduce ethanol consumption was diminished in β3-S408A/S409A mice. Our results provide strong evidence that PKA-dependent phosphorylation of β3 GABAA receptor subunits is an important mechanism by which apremilast increases acute sensitivity to alcohol, decreases AFT, and decreases ethanol drinking.

Limonium tetragonum Promotes Running Endurance in Mice through Mitochondrial Biogenesis and Oxidative Fiber Formation.

The purpose of this study was to examine whether Limonium tetragonum, cultivated in a smart-farming system with LED lamps, could increase exercise capacity in mice. C57BL/6 male mice were orally administered vehicle or Limonium tetragonum water extract (LTE), either 30 or 100 mg/kg, and were subjected to moderate intensity treadmill exercise for 4 weeks. Running distance markedly increased in the LTE group (100 mg/kg) by 80 ± 4% compared to the vehicle group, which was accompanied by a higher proportion of oxidative fibers (6 ± 6% vs. 10 ± 4%). Mitochondrial DNA content and gene expressions related to mitochondrial biogenesis were significantly increased in LTE-supplemented gastrocnemius muscles. At the molecular level, the expression of PGC-1α, a master regulator of fast-to-slow fiber-type transition, was increased downstream of the PKA/CREB signaling pathway. LTE induction of the PKA/CREB signaling pathway was also observed in C2C12 cells, which was effectively suppressed by PKA inhibitors H89 and Rp-cAMP. Altogether, these findings indicate that LTE treatment enhanced endurance exercise capacity via an improvement in mitochondrial biosynthesis and the increases in the formation of oxidative slow-twitch fibers. Future study is warranted to validate the exercise-enhancing effect of LTE in the human.

Paclitaxel Regulates TRPA1 Function and Expression Through PKA and PKC

Paclitaxel (PTX) is a frequently used anticancer drug that causes peripheral neuropathy. Transient receptor potential ankyrin 1 (TRPA1), a plasma membrane calcium channel, has been associated with PTX toxicity and with other chemotherapy agents such as oxaliplatin and vincristine. However, the effect of PTX on the functional expression and calcium currents of TRPA1 has not been determined. The present study shows the effect of PTX on TRPA1 activity in a neuronal cell line (SH-SY5Y). The effect of PTX on the expression of TRPA1 was assessed through quantitative PCR and Western blot analyses to determine the relative mRNA and protein expression levels. To assess the effect on calcium flux and currents, cells were exposed to PTX; simultaneously, a specific agonist and antagonist of TRPA1 were added to evaluate the differential response in exposed versus control cells. To assess the effect of PKA, PKC and PI3K on PTX-induced TRPA1 increased activity, selective inhibitors were added to these previous experiments. PTX increased the mRNA and protein expression of TRPA1 as well as the TRPA1-mediated Ca2+ currents and intracellular Ca2+ concentrations. This effect was dependent on AITC (a selective specific agonist) and was abolished with HC-030031 (a selective specific antagonist). The inhibition of PKA and PKC reduced the effect of PTX on the functional expression of TRPA1, whereas the inhibition of PI3K had no effects. PTX-induced neuropathy involves TRPA1 activity through an increase in functional expression and is regulated by PKA and PKC signaling. These findings support the role of the TRPA1 channel in the mechanisms altered by PTX, which can be involved in the process that lead to chemotherapy-induced neuropathy.

Multiple tachykinins and their receptors characterized in the gastropod mollusk Pacific abalone: Expression, signaling cascades, and potential role in regulating lipid metabolism.

Tachykinin (TK) families, including the first neuropeptide substance P, have been intensively explored in bilaterians. Knowledge of signaling of TK receptors (TKRs) has enabled the comprehension of diverse physiological processes. However, TK signaling systems are largely unknown in Lophotrochozoa. This study identified two TK precursors and two TKR isoforms in the Pacific abalone Haliotis discus hannai (Hdh), and characterized Hdh-TK signaling. Hdh-TK peptides harbored protostomian TK-specific FXGXRamide or unique YXGXRamide motifs at the C-termini. A phylogenetic analysis showed that lophotrochozoan TKRs, including Hdh-TKRs, form a monophyletic group distinct from arthropod TKRs and natalisin receptor groups. Although reporter assays demonstrated that all examined Hdh-TK peptides activate intracellular cAMP accumulation and Ca2+ mobilization in Hdh-TKR-expressing mammalian cells, Hdh-TK peptides with N-terminal aromatic residues and C-terminal FXGXRamide motifs were more active than shorter or less aromatic Hdh-TK peptides with a C-terminal YXGXRamide. In addition, we showed that ligand-stimulated Hdh-TKRs mediate ERK1/2 phosphorylation in HEK293 cells and that ERK1/2 phosphorylation is inhibited by PKA and PKC inhibitors. In three-dimensional in silico Hdh-TKR binding modeling, higher docking scores of Hdh-TK peptides were consistent with the lower EC50 values in the reporter assays. The transcripts for Hdh-TK precursors and Hdh-TKR were highly expressed in the neural ganglia, with lower expression levels in peripheral tissues. When abalone were starved for 3 weeks, Hdh-TK1 transcript levels, but not Hdh-TK2, were increased in the cerebral ganglia (CG), intestine, and hepatopancreas, contrasting with the decreased lipid content and transcript levels of sterol regulatory element-binding protein (SREBP). At 24 h post-injection in vivo, the lower dose of Hdh-TK1 mixture increased SREBP transcript levels in the CG and hepatopancreas and accumulative food consumption of abalone. Higher doses of Hdh-TK1 and Hdh-TK2 mixtures decreased the SREBP levels in the CG. When Hdh-TK2-specific siRNA was injected into abalone, intestinal SREBP levels were significantly increased, whereas administration of both Hdh-TK1 and Hdh-TK2 siRNA led to decreased SREBP expression in the CG. Collectively, our results demonstrate the first TK signaling system in gastropod mollusks and suggest a possible role for TK peptides in regulating lipid metabolism in the neural and peripheral tissues of abalone.

Discovery of Pinostrobin as a Melanogenic Agent in cAMP/PKA and p38 MAPK Signaling Pathway.

Melanogenesis is the process of melanin synthesis to protect the skin against ultraviolet radiation and other external stresses. The loss of skin pigmentation is closely related to depigmented skin disorders. The melanogenic effects of pinostrobin, an active flavanone found in honey, were evaluated. B16F10 cells were used for melanin content, tyrosinase activity, and the expression of melanogenesis-related markers. Moreover, computational simulations were performed to predict docking and pharmacokinetics. Pinostrobin increased melanin levels and tyrosinase activity by stimulating the expression of melanogenic regulatory factors including tyrosinase, tyrosinase-related protein (TRP) 1 and microphthalmia transcription factor (MITF). Specifically, the phosphorylation of cAMP response element binding (CREB) involved in the MITF activation was augmented by pinostrobin. Moreover, the compound upregulated the β-catenin by cAMP/PKA-mediated GSK-3β inactivation. Co-treatment with a PKA inhibitor, inhibited melanin production, tyrosinase activity, and expression of MITF, p-CREB, p-GSK-3β and p-β-catenin, demonstrating that pinostrobin-stimulated melanogenesis was closely related to cAMP/PKA signaling pathway. Furthermore, the combination of pinostrobin and a specific p38 inhibitor, showed that MITF upregulation by pinostrobin was partly associated with the p38 signaling pathway. Docking simulation exhibited that the oxygen group at C-4 and the hydroxyl group at C-5 of pinostrobin may play an essential role in melanogenesis. In silico analysis revealed that pinostrobin had the optimal pharmacokinetic profiles including gastrointestinal absorption, skin permeability, and inhibition of cytochrome (CYP) enzymes. From the present results, it might be suggested that pinostrobin could be useful as a potent and safe melanogenic agent in the depigmentation disorder, vitiligo.

Astragaloside IV ameliorates cerebral ischemia-reperfusion injury via upregulation of PKA and Cx36.

To determine the effects of astragaloside IV on cerebral ischemic-reperfusion injury in rats and to explore underlying mechanisms of brain protection. Sixty Sprague-Dawley rats were randomized into four groups: Sham, cerebral ischemia-reperfusion (I/R group), I/R+astragaloside IV (I/R+AST-IV group) and I/R+astragaloside IV+PKA kinase inhibitor H-89 (I/R+AST-IV+H-89 group). All I/R rats were subjected to 2 h cerebral ischemia, followed by 24 h reperfusion and scored for neurobehavior. Cerebral infarct volume, pathomorphological changes and brain apoptosis, in addition to changes in expression of Cx36, PKA, Bax and Bcl-2 proteins, were assessed. Astragaloside IV treatment reduced neurobehavioral score and percentage volume of cerebral infarct, reducing pathomorphological injury and brain apoptosis. Expressions of Cx36 and PKA protein were increased and the Bax/Bcl-2 ratio decreased. All astragaloside IV effects were reversed by the PKA inhibitor and H-89. Astragaloside IV attenuated cerebral I/R injury in rats by increasing Cx36 and PKA protein expression and reducing the Bax/Bcl-2 ratio.

A2A R mediated modulation in IP3 levels altering the [Ca2+]i through cAMP-dependent PKA signalling pathway

Stimulation of A2A receptors (A2A R) coupled to Gs/olf protein activates Adenylyl cyclase (AC) leading to the release of cAMP which activates the cAMP-dependent PKA phosphorylation. The possible role of A2A R in the modulation of free cytosolic Ca2+ concentration ([Ca2+]i) involving IP3, cAMP and PKA was investigated in HEK 293-A2A R. The levels of IP3 and cAMP were observed by enzyme immunoassay detection method and [Ca2+]i using Fluo-4 AM. Moreover, cAMP-dependent PKA was determined using the PKA Colorimetric Activity Kit. We observed that the cells pre-treated with A2A R agonist NECA showed increased levels of cAMP, PKA, IP3 and [Ca2+]i levels. However, the reverse effect was observed with A2A R antagonists (ZM241385 and caffeine). Blocking the Gαq/PLC/DAG/IP3 pathway with neomycin, a PLC inhibitor did not affect the modulation of IP3 and [Ca2+]i levels in HEK 293-A2A R cells. To investigate the Gαi/AC/cAMP/PKA, HEK 293-A2A R cells pre-treated with pertussis toxin followed by forskolin in the presence of A2A R agonist (NECA) showed no effect on cAMP levels. Further, Gαs/AC/cAMP/PKA pathway was investigated to elucidate the role of cAMP-dependent PKA in IP3 mediated [Ca2+]i modulation. In the HEK 293-A2A R cells pre-treated with PKA inhibitor KT5720 and treated with NECA led to inhibit the IP3 and [Ca2+]i levels. The study distinctly demonstrated that A2A R modulates IP3 levels to release the [Ca2+]i via cAMP-dependent PKA. The role of A2A R mediated Gαs pathway inducing IP3 mediated [Ca2+]i release may open new avenues in the therapy of neurodegenerative disorder.

Hyperoside improves learning and memory deficits by amyloid β1-42 in mice through regulating synaptic calcium-permeable AMPA receptors

Alzheimer's disease (AD) is the most common degenerative disease and is indicative of dementia. The cerebral accumulation of amyloid β (Aβ), a crucial factor in AD, initiates synaptic and cognitive dysfunction. Therefore, the elevation of synaptic and cognitive functions may help manage dementia in AD. In this study, we suggest hyperoside as a synaptic function- and memory-enhancing agent. Hyperoside enhanced learning and memory in passive avoidance and object recognition tasks. Hyperoside facilitated synaptic long-term potentiation (LTP) in acute hippocampal slices. IEM-1460, a calcium-permeable amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (CP-AMPAR) antagonist, blocked the facilitation effect of hyperoside. Hyperoside also induced N-methyl-d-aspartate receptor (NMDAR)-independent LTP, which was blocked by IEM-1460, suggesting the involvement of CP-AMPARs in the synaptic effects of hyperoside-mediated LTP. PKI (a PKA inhibitor) or SQ22536 (adenylyl cyclase, an AC inhibitor) blocked hyperoside-facilitated LTP and hyperoside-induced NMDAR-independent LTP. Hyperoside-enhanced learning and memory were blocked by IEM-1460, suggesting the involvement of CP-AMPARs in the effect of hyperoside on learning and memory. Finally, hyperoside ameliorated Aβ-induced memory impairments in an AD mouse model. These results suggest that hyperoside enhances learning and memory, and this may be due to the effect of CP-AMPARs.

Cryptochrome 1 is involved in anti-bacterial immunity in Japanese flounder (Paralichthys olivaceus)

Cryptochrome is a pivotal protein of the core regulatory machinery in circadian clock and participates in various biological processes including immunity. In fish, the immunological functionality of Cryptochrome is unclear. In this study, we profiled the expression pattern of Japanese flounder cryptochrome 1 (named PoCry1) and investigated its involvement in the immune response during bacterial infection. PoCry1 is approximately 75% identical in amino acid sequence to mammalian Cryptochrome 1 and possesses the conserved DNA photolyase domain and FAD binding domain. Its transcription was detected in 10 flounder tissues and was reduced upon Vibrio harveyi challenge. The transcription of PoCry1 exhibited apparent rhythmic oscillation upon the light actuation of a day-night cycle in different tissues. The knockdown of PoCry1 resulted in significant augment of inflammatory cytokine expression. During bacterial infection, PoCry1 silencing led to increase in cytokine expression and decrease in bacterial dissemination, which could be neutralized by a specific PKA inhibitor, suggesting PoCry1 is likely able to negatively modulate the immune response to bacterial infection through PKA associated pathways.Taken together, our results revealed that PoCry1 is circadian oscillated in transcription upon rhythmic light stimulation and involved in anti-infection immunity, thus enlightening that light condition as a circadian zeitgeber could be exploited to strength fish immune system and benefit disease control in aquaculture.

Islet MC4R Regulates PC1/3 to Improve Insulin Secretion in T2DM Mice via the cAMP and β-arrestin-1 Pathways.

Melanocortin-4 receptor (MC4R) plays an important role in energy balance regulation and insulin secretion. It has been demonstrated that in the pancreas, it is expressed in islet α and β cells, wherein it is significantly correlated with insulin and glucagon-like peptide-1 (GLP-1) secretion. However, the molecular mechanism by which it regulates islet function is still unclear. Therefore, in this study, our aim was to clarify the signaling and target genes involved in the regulation of insulin and GLP-1 secretion by islet MC4R. The results obtained showed that in islet cells, the expression of prohormone convertase 1/3 (PC1/3), which is correlated with islet GLP-1 and insulin secretion, increased significantly under the action of the MC4R agonist, NDP-α-MSH, but decreased under the action of the MC4R antagonist, AgRP. Additionally, we observed that to exert their regulatory functions in the islets, cAMP and β-arrestin-1 acted as important signaling mediators of MC4R, and compared with control islets, the cAMP, PKA, and β-arrestin-1 levels corresponding to NDP-α-MSH-treated islets were significantly elevated; however, in AgRP-treated islets, their levels decreased significantly. Islets treated with the PKA inhibitor, H89, and the ERK1/2 inhibitor, PD98059, also showed significant decreases in PC1/3 expression level, indicating that the cAMP and β-arrestin-1 pathways are significantly correlated with PC1/3 expression. These findings suggest that islet MC4R possibly affects PC1/3 expression via the cAMP and β-arrestin-1 pathways to regulate GLP-1 and insulin secretion. These results provide a new theoretical basis for targeting the molecular mechanism of type 2 diabetes mellitus.

Oncogene addiction to GNAS in GNASR201 mutant tumors.

The GNASR201 gain-of-function mutation is the single most frequent cancer-causing mutation across all heterotrimeric G proteins, driving oncogenesis in various low-grade/benign gastrointestinal and pancreatic tumors. In this study, we investigated the role of GNAS and its product Gαs in tumor progression using peritoneal models of colorectal cancer (CRC). GNAS was knocked out in multiple CRC cell lines harboring GNASR201C/H mutations (KM12, SNU175, SKCO1), leading to decreased cell-growth in 2D and 3D organoid models. Nude mice were peritoneally injected with GNAS-knockout KM12 cells, leading to a decrease in tumor growth and drastically improved survival at 7 weeks. Supporting these findings, GNAS overexpression in LS174T cells led to increased cell-growth in 2D and 3D organoid models, and increased tumor growth in PDX mouse models. GNAS knockout decreased levels of cyclic AMP in KM12 cells, and molecular profiling identified phosphorylation of β-catenin and activation of its targets as critical downstream effects of mutant GNAS signaling. Supporting these findings, chemical inhibition of both PKA and β-catenin reduced growth of GNAS mutant organoids. Our findings demonstrate oncogene addiction to GNAS in peritoneal models of GNASR201C/H tumors, which signal through the cAMP/PKA and Wnt/β-catenin pathways. Thus, GNAS and its downstream mediators are promising therapeutic targets for GNAS mutant tumors.

α and β catalytic subunits of cAMP‐dependent protein kinase regulate formoterol‐induced inflammatory gene expression changes in human bronchial epithelial cells

It has been proposed that genomic mechanisms contribute to adverse effects often experienced by asthmatic subjects who take regular, inhaled β2 -adrenoceptor agonists as a monotherapy. Moreover, data from preclinical models of asthma suggest that these gene expression changes are mediated by β-arrestin-2 rather than PKA. Herein, we tested this hypothesis by comparing the genomic effects of formoterol, a β2 -adrenoceptor agonist, with forskolin in human primary bronchial epithelial cells (HBEC). Gene expression changes were determined by RNA-sequencing. Gene silencing and genome editing were employed to explore the roles of β-arrestin-2 and PKA. The formoterol-regulated transcriptome in HBEC treated concurrently with TNFα was defined by 1480 unique gene expression changes. TNFα-induced transcripts modulated by formoterol were annotated with enriched gene ontology terms related to inflammation and proliferation, notably "GO:0070374~positive regulation of ERK1 and ERK2 cascade," which is an apparentβ-arrestin-2 target. However, expression of the formoterol- and forskolin-regulated transcriptomes were highly rank-order correlated and the effects of formoterol on TNFα-induced inflammatory genes were abolished by an inhibitor of PKA. Furthermore, formoterol-induced gene expression changes in BEAS-2B bronchial epithelial cell clones deficient in β-arrestin-2 were comparable with those expressed by their parental counterparts. Contrariwise, gene expression was partially inhibited in clones lacking the α-catalytic subunit (Cα) of PKA and abolished following the additional knockdown of the β-catalytic subunit (Cβ) paralogue. The effects of formoterol on inflammatory gene expression in airway epithelia are mediated by PKA and involve the cooperation of PKA-Cα and PKA-Cβ.

P-077 Human sperm acrosome becomes alkalinized during capacitation

Abstract Study question Does during the human sperm capacitation the acrosomal pH (pHa) increases naturally and how it change can be modulate? Summary answer Sperm acrosome is progressively alkalinized during capacitation in a HCO3- and Ca2+ dependent manner. pHa is regulated by V-ATPase and involving the sAC-PKA signaling pathway. What is known already Capacitation involves biochemical and physiological modifications undergone by sperm as they travel through the female reproductive tract. These modifications prepare the sperm to enact the acrosome reaction (AR), an essential step for egg fertilization. Capacitation requires [Ca2+]i and pHi increases. Mouse sperm capacitation is accompanied by acrosomal alkalinization and artificial elevation of the pHa is sufficient to trigger the AR in mouse and human sperm, but it is unknown if the pHa increases physiologically during human sperm capacitation. Study design, size, duration In this work we followed pHa changes during human sperm capacitation (6 h) and evaluated the mechanisms of its regulation using pharmacological tools. Sperm were stained with LysoSensor green and the relative fluorescence measurements for pHa kinetics during capacitation were performed at 15 min of incubation (initial time: 0 h) and after 3 and 6 h of incubation. Independent experiments of at least 3 donors were performed per condition. Participants/materials, setting, methods We used semen samples from 11 healthy donors fulfilled the requirements defined by the OMS (OMS, 2010). Sperm recovered by swim-up were incubated in a capacitating or non-capacitating HTF medium. LysoSensor green fluorescence dye was used to evaluate pHa dynamic during capacitation using single cell epifluorescence microscopy and image-based flow cytometry. Additionally, pHa was evaluated in the presence of specific inhibitors to assess regulatory mechanisms. A linear model was used to study the pHa kinetics. Main results and the role of chance Human sperm pHa is alkalinized progressively at 3 and 6 h of the capacitation (p < 0.05). V-ATPase activity mainly responsible for the acrosome acidity and can modulate at least in part acrosome alkalinization during capacitation (***p<0.001, *p<0.05). V-ATPase is immunolocalized to the acrosome and equatorial segment of human sperm. Bicarbonate (HCO3-) was essential for the pHa increase observed during capacitation and its absence inhibits completely the acrosome alkalinization (**p<0.01, *p<0.05). Inhibition of the Cystic Fibrosis Transmembrane Regulator (CFTR) or the Na+/HCO3- Cotransporter (NBC) partially blocked the pHa changes during capacitation. Extracelular Ca2+ is involved in pHa regulation during capacitation. Under low external Ca2+ the pHa regulation is altered and occurred delay in acrosome alkalinization (*p < 0.05 and **p < 0.001). Finally, we tested a pharmacological blockade of sAC and PKA during capacitation. sAC inhibition did not prevent acrosome alkalinization, but it significantly reduced the rate of the pHa changes as compared to the capacitation control (***p=0.0009), revealing a certain influence. On the other hand, PKA inhibition induced acrosomal acidification at the start of capacitation and accelerated significantly the alkalinization (*p<0.05). Together these results allow us to assume that a signaling pathway mediated to sAC is participating in the pHa regulation. Limitations, reasons for caution Experiments were performed in vitro and due to model limitations is difficult to establish strategies that allow us to know more directly, other molecular mechanisms that regulate the pHa. Wider implications of the findings Our findings show that pHa is subject of regulation during human sperm capacitation. The results provide new insights regarding the molecular mechanisms involved in human sperm capacitation and this knowledge results interesting for the study of specific molecules that inhibit capacitation or AR. Trial registration number Not applicable

OLHA (Nα-oleoylhistamine) modulates activity of mouse brain histaminergic neurons

Histaminergic (HA) neurons are located in the tuberomamillary nucleus (TMN) of the posterior hypothalamus, from where they project throughout the whole brain to control wakefulness. We examined the effects of Nα-oleoylhistamine (OLHA), a non-enzymatic condensation product of oleic acid (OLA) and histamine, on activity of mouse HA neurons in brain slices. OLHA bidirectionally modulated the firing of HA neurons. At 10 nM OLHA inhibited or had no action, whereas at 1 μM it evoked excitatory and inhibitory responses. Inhibition was not seen in presence of the histamine receptor H3 (H3R) antagonist clobenpropit and in calcium-free medium. Pre-incubation with a histamine-reuptake blocker prevented the decrease in firing by OLHA. OLHA-evoked increase in firing (EC50 ∼44 nM) was insensitive to blockers of cannabinoid 1 and 2 receptors and of the capsaicin receptor, but was significantly impaired by the peroxisome proliferator-activated receptor-alpha (PPAR-alpha) antagonist MK886, which suppressed also the rise in intracellular calcium level caused by OLHA. The OLHA-evoked excitation was mimicked by synthetic PPAR-alpha agonists (gemfibrozil and GW7647) and was abolished by the PKA inhibitor H-89. The H3R affinity (Ki) for histamine, measured in HEK293 cells with stable expression of human H3R, was higher than for OLHA (Ki: 42 vs 310 nM, respectively). Expression of PPAR-alpha was not different between TMN regions of males and females, responses to OLHA did not differ. Molecular modelling of PPAR-alpha bound to either OLHA or OEA showed similar binding energies. These findings shed light on a novel biotransformation product of histamine which may play a role in health and disease.

Long-Acting β2 Adrenergic Receptor Agonist Ameliorates Imiquimod-Induced Psoriasis-Like Skin Lesion by Regulating Keratinocyte Proliferation and Apoptosis.

Psoriasis is a chronic inflammatory disease that affects approximately 1%–5% of the population worldwide. Considering frequent relapse, adverse drug reactions, and large costs of treatment, it is urgent to identify new medications for psoriasis. Keratinocytes play an essential role during psoriasis development, and they express high levels of β2-Adrenergic receptor (β2-AR), which increases intracellular cAMP levels when activated. Increased level of cAMP is associated with the inhibition of epidermal cell proliferation. In the present study, we observed the effect of salmeterol, a long-acting β2-AR agonist, on the proliferation and apoptosis of keratinocytes as well as imiquimod-induced psoriasis-like skin lesions in mice. As phosphodiesterase 4 (PDE4) inhibitors increases intracellular cAMP concentration by inhibiting its inactivation, we further explored the synergetic effect of a PDE4 inhibitor and salmeterol on psoriasis-like skin lesions in mice. Our results indicated that salmeterol effectively inhibited the proliferation of HaCaT cells induced by TNF-α and serum, and this effect was accompanied by significantly increased apoptosis and CREB phosphorylation, which were reversed by the PKA inhibitor, H89. Salmeterol ameliorated imiquimod-induced psoriasis-like skin lesions in mice, but salmeterol combined with a PDE4 inhibitor had no synergetic effect in improving skin lesions in mice. Of note, the synergistic effects of anti-proliferation and induction of apoptosis in HaCaT cells appeared by inhibiting ERK signaling. In summary, salmeterol, a long-acting β2-AR agonist, alleviates the severity of psoriasis via inhibiting the proliferation and promoting apoptosis of keratinocytes, partially by activating the cAMP/PKA signaling pathway.

Abstract 822: Oncogene addiction to GNAS in GNASR201 mutant tumors

Abstract Background: The GNASR201 mutation is the single most frequent cancer-causing mutation across all heterotrimeric G proteins. This gain of function mutation in GNAS drives oncogenesis in appendiceal, colorectal, and gastric adenocarcinoma, as well as Intraductal Papillary Mucinous Neoplasms (IPMN) and Small Cell Lung Cancer (SCLC). In this study, we investigated the role of GNAS in tumor growth using peritoneal models of colorectal cancer (CRC). Methods: GNAS was knocked out in multiple GNASR201C/H mutant colon cell lines (KM12, SNU175, and SKCO1) and overexpressed in GNASWT LS174T cells. Isogenic pairs of KM12 and LS174T cells were injected into the peritoneum of NSG mice to study the role of GNAS in cell line derived xenograft (CDX) models of peritoneal metastasis. Cell lines and CDX were profiled with RNAseq, reverse phase protein assay (RPPA), and immunohistochemistry (IHC) to identify potential pathways regulated by mutant GNAS. Identified mediators of GNAS signaling were then validated using chemical inhibitors of PKA (H-89) and β-catenin (LF3). Results: GNAS knockout significantly decreased 2D colony formation by KM12 (68%), SNU175 (76%), and SKCO1 (85%) cells (all p < 0.0001), and decreased 3D organoid area of KM12 cells by 62% (p = 0.043). There was significant increase in colony formation by LS174T cells overexpressing GNASR201C (193%, p = 0.016) and GNASR201H (170%, p =0.0037). Mice injected with KM12 GNAS-knockout tumors exhibited a significant 68% reduction in tumor growth (n = 6, p = 0.016) and were more likely to survive at 7 weeks (0% vs. 100%, p = 0.0007) relative to those with parent KM12-GNASR201H tumors. Likewise, mice injected with GNASR201H LS174T cells showed significant increase in tumor growth (934% vs. 100%, n = 3, p = 0.042). Histology of GNAS-knockout tumors showed a marked decrease in mucinous stroma and increased lytic necrosis, suggesting an interaction between oncogenic GNAS signaling and the peritoneal environment. GNAS is known to stimulate adenylate cyclase and GNAS knockout decreased levels of cyclic AMP in KM12 cells, confirming an on-target effect. RPPA and RNAseq profiling of KM12 and LS174T PDX tumors identified phosphorylation of β-catenin and activation of Wnt/β-catenin targets (NES = 1.25, 1.24) as critical downstream effects of mutant GNAS signaling, confirmed by a 4.9-fold (p = 0.001) increase in nuclear β-catenin intensity in LS174T GNASR201H tumors (19% vs. 99% nuclei stained positive, p = 0.0002). Chemical inhibition of both PKA and β-catenin reduced growth of GNAS mutant organoids by 79% (p = 0.004) and 67% (p = 0.007) respectively, supporting the involvement of the cAMP/PKA and β-catenin pathways in mutant GNAS signaling. Conclusions: Our findings demonstrate oncogene addiction to GNAS in peritoneal models of GNASR201C/H tumors, which signal through the cAMP/PKA and Wnt/β-catenin pathways. Thus, GNAS and its downstream mediators are promising therapeutic targets for GNAS mutant tumors. Citation Format: Aditya More, Valsala Haridas, Ichiaki Ito, Saikat Chowdhury, Yue Gu, Natalie W. Fowlkes, John P. Shen. Oncogene addiction to GNAS in GNASR201 mutant tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 822.

Genistein accelerates glucose catabolism via activation the GPER-mediated cAMP/PKA-AMPK signaling pathway in broiler chickens

Genistein, the most abundance of phytoestrogens in soybeans, has beneficial effects in regulating metabolism-related disease; however, there is few available literatures about whether genistein regulates glucose metabolism that in turn affects the lipid accumulation in animals or humans. The current study showed that genistein promoted glucose uptake by enhancing glucose transporter-2 (GLUT2) protein level; and it also increased the activity of phosphofructokinase-1 (PFK) and pyruvate dehydrogenase (PDH), and the mRNA level of succinate dehydrogenase (SDH) both in broiler chickens or hepatocytes. Moreover, genistein obviously increased the p-LKB1 and p-AMPKα protein levels both in vivo and in vitro. Furthermore, the enhancement of genistein on glucose uptake and catabolism were reversed in hepatocytes pre-treated with AMPK inhibitor Compound C, and the increasing of genistein on the p-LKB1 and p-AMPKα protein levels were also reversed in hepatocytes pre-treated with PKA inhibitor H89. Importantly, the results showed that genistein simultaneously increased the estrogen receptor β (ERβ) and G protein-coupled estrogen receptor (GPER) protein levels, but the elevation effect of genistein on cAMP content was completely reversed in hepatocytes pre-treated with GPER antagonist G15, rather than ERβ inhibitor PHTPP. Meanwhile, the increasing of p-LKB1 and p-AMPKα protein levels induced by genistein were also reversed in hepatocytes pre-treated with G15. Collectively, our data demonstrated that genistein improves glucose metabolism via activating the GPER-mediated cAMP/PKA-AMPK signaling pathway. These findings provide theoretical basis for genistein as a promising nutritional supplemental to alleviate metabolism disorders and related diseases in animals or even humans.

Hypothalamic response with PKA/CREB signaling is associated with direct cerebroventricular administration of bombesin-induced scratching

Bombesin (BN) is an itch-specific mediator that causes intense itch-scratching activity in mammals. Although most examinations of BN-induced itch processing have focused on the spinal cord, the involvement of central nervous system mechanisms remains unclear. Here, we investigated how relationships among hypothalamic regions regulate BN-mediated itch-scratch processes. We found that intracerebroventricular (i.c.v.) administration of BN (0.04–4 μg) elicited intense itch scratching in mice, whereas BN (0.4–400 μg) administered via intravenous tail injection failed to evoke a scratching response. Additionally, nalfurafine had no significant effects on BN-induced scratching behavior, indicating that central modulation of BN is distinct from histamine-mediated histaminergic itch and chloroquine-mediated non-histaminergic itch signaling pathways. We labeled BN with a fluorescent tag, 7-nitrobenz-2-oxa-1 (NBD), and traced its fluorescence in the hypothalamus for 30 min following i.c.v. NBD-BN administration. Accordingly, we confirmed that i.c.v. administration of BN enhanced c-Fos expression in the dorsal medial nucleus of the hypothalamus, where neuromedin B receptors and gastrin-releasing peptide receptors are highly expressed. Interestingly, in situ injection of BN into the hypothalamus immediately and robustly induced itch-scratching behavior. Moreover, gene transcripts and western blot assay revealed that BN receptor-dependent PKA/CREB signaling was upregulated in the hypothalamus after i.c.v. administration of BN. Consistently, pretreatment with a PKA inhibitor, Rp-cAMP, significantly reduced BN-induced scratching behavior. Our results indicate that the dorsal medial nucleus of the hypothalamus may be a key nucleus in mediating BN-mediated itch and hypothalamic PKA/CREB signaling is involved in regulating BN-mediated itch.

PDE4D binds and interacts with YAP to cooperatively promote HCC progression

The role of cAMP in the development of hepatocellular carcinoma (HCC) is controversial and the biological function of cAMP-hydrolysing enzyme phosphodiesterase 4D (PDE4D) in HCC remains unclear. In this study, we observed markedly higher PDE4D expression in HCC patients with poor survival. PDE4D bound to yes-associated protein (YAP), and PDE4D expression positively correlated with YAP expression in HCC. Overexpression of PDE4D increased YAP dephosphorylation and activity and promoted HCC cell growth in vitro and in vivo, which was attenuated by the YAP inhibitor verteporfin. In contrast, silencing PDE4D reduced YAP expression and HCC cell growth. Notably, forced expression of YAP promoted PDE4D and YAP target gene expression and cell growth, which were abrogated by the PDE4D inhibitor roflumilast. Mechanistically, silencing of YAP caused PDE4D downregulation and HCC cell apoptosis via extracellular signal-regulated kinase (ERK) activation. Roflumilast activated cAMP-PKA signaling and induced cAMP-PKA-dependent YAP phosphorylation at serine 127, resulting in YAP degradation and suppression of HCC growth, which were reversed by the PKA inhibitor PKI. Additionally, transfection of the YAP-S127A mutant reversed roflumilast-mediated suppression of YAP and cell growth. Taken together, our findings indicate that PDE4D binds to and interacts with YAP to promote HCC progression. Targeting the PDE4D-YAP interaction with roflumilast may be an effective strategy for HCC treatment.

Memory-related Induction Mechanisms Trigger Persistent Hyperexcitability of Nociceptors

Persistent hyperexcitability of primary nociceptors is a major contributor to diverse forms of chronic pain. While much is known about mechanisms that maintain persistent hyperexcitability, little is known about induction mechanisms. Interesting parallels have been noted between processes that can induce long-lasting nociceptor hyperexcitability and mechanisms involved in memory formation (e.g., Weragoda et al., 2004; J Neurosci 24:10393; Price et al., 2015, Prog Mol Biol Transl Sci, 131:409). We have developed an in vitro model to test the hypothesis that cellular processes important for the induction of late long-term synaptic potentiation (L-LTP) and memory in the hippocampus can also induce long-term hyperexcitability (LTH) of dissociated rat nociceptor somata. We have begun by testing whether nociceptor LTH can be induced by cAMP signaling involving PKA activity, CREB activity, gene transcription, and protein synthesis. Rat DRG neurons were dissociated and cultured overnight. Beginning 1 h after dissociation, neurons were incubated for 6 h with the adenylyl cyclase activator, forskolin, or a Gs-coupled serotonin receptor (5-HT4R) agonist, prucalopride. Other neurons received co-treatment with either a PKA inhibitor, H89; a CREB inhibitor, 666-15; a transcription inhibitor, actinomycin D; or a translation inhibitor, cycloheximide, at concentrations known to block the induction of hippocampal L-LTP. Whole-cell patch recordings under current-clamp were made 12-24 h after washout. The early treatment with forskolin or prucalopride induced LTH manifested as depolarization of resting membrane potential and increased action potential generation when neurons were further depolarized artificially to -45 mV for 30 s. Co-treatment during induction by prucalopride with H89, 666-15, actinomycin D, or cycloheximide significantly reduced LTH. These results support the hypothesis that conserved, memory-related mechanisms can induce nociceptor alterations known to promote persistent pain. This preparation enables efficient, detailed examination of numerous potential induction mechanisms, and might reveal new pharmacological targets for preventing the transition to chronic pain. Grant support from NIH grants NS091759 and NS111521.