Protein Kinase A Signaling Pathway Research Articles

The disrupting effect of chlormequat chloride on growth hormone is associated with pregnancy

Since chlormequat chloride is widely applied as a plant growth regulator in agriculture and horticulture, its exposure through food consumption is common. We demonstrated previously that chlormequat chloride exposure during pregnancy led to embryos with bigger sizes associated with higher levels of growth hormone (GH) on gestation day 11 (GD11). However, the dose-effect relationship of chlormequat chloride at a lower dose range was not established, and the underlying mechanisms of its promoting effects on embryonic growth and development were not fully elucidated. To address these, pregnant rats were orally exposed to chlormequat chloride at 0, 0.05, 0.5 and 5 mg/kg.bw from GD0 to 11 and the embryonic growth and growth related hormones were evaluated on GD11. We found that the growth and development of the embryos was significantly promoted in a dose dependent manner by chlormequat chloride. Chlormequat chloride also increased embryonic GH, GH releasing hormone (GHRH), and somatostatin (SRIF), and inhibited the embryonic cAMP dependent protein kinase A (PKA) signaling pathway. Chlormequat chloride increased GH synthesis modulated by GHRH/SRIF-PKA-Pituitary specific transcription factor 1 (Pit-1) in the maternal rats. Intriguingly, chlormequat chloride did not show any effects on GH and PKA signaling pathways in the non-pregnant female rats. These findings together suggest that the disrupting effect of chlormequat chloride on GH is associated with pregnancy.

The role of protein kinase A signaling in cardiomyopathy

Cardiomyopathy, a diverse group of heart muscle diseases, poses a significant challenge to global cardiovascular health. However, the intricate molecular mechanisms underlying cardiomyopathy, especially the pivotal role of protein kinase A (PKA) signaling pathways in its pathogenesis, remains unclear. The review synthesizes current research findings and highlights the multifaceted interactions between PKA and key cellular components implicated in cardiomyopathy progression. Moreover, this paper unfolds with an exploration of the normal physiological functions of PKA in cardiac cells, emphasizing its role in regulating crucial processes such as excitation-contraction coupling and cellular energy homeostasis. The article navigates through the dysregulation of PKA signaling observed in various forms of cardiomyopathy, including hypertrophic, dilated, and restrictive subtypes. Insightful analyses into the impact of PKA-mediated phosphorylation events on cardiac contractility, ion channel function, and mitochondrial dynamics shed light on the intricate interplay between signaling cascades and disease manifestation. Future clinical trials targeting the PKA signaling pathway are required to seek more effective and precise therapeutic approaches of cardiomyopathy.

Attenuation of propofol-induced hippocampal neuron damage in developing rats by dexmedetomidine

This study explored the mechanism of dexmedetomidine attenuating propofol damage to hippocampal neurons in rats. By constructing rat hippocampal neuron model and carrying out targeted intervention; enzyme linked immunosorbent assay (ELISA) detected IL-1β and IL-18 levels; while Nissl staining observed hippocampus Histopathology; flow cytometry quantitatively analyzed the number of hippocampal neuron cells and apoptosis rate. Moreover, levels of PKA, Caspase-1 and NOD-like receptor thermal protein domain associated protein 3(NLRP3) and corresponding proteins were measured. Results showed that, there were more Nissl bodies in cytoplasm of hippocampal neurons in control group, and distribution in the cytoplasm was relatively uniform. Moreover, IL-1β and IL-18 in model group continued to increase; while dexmedetomidine effectively inhibited apoptosis of hippocampal neurons, which may be related to decreased expression of protein kinase A (PKA). After using PKA inhibitors, apoptosis was significantly inhibited, and when the expression of PKA was reduced, Caspase-1 was down-regulated along with reduced NLRP3 level, which improved the injury of hippocampal neurons. Dexmedetomidine can therefore down-regulate the level of Caspase-1 in hippocampal neurons by inhibiting the PKA signaling pathway, improving apoptosis, reducing the genetic and protein expressions of NLRP3, and slowing down the damage of hippocampal neurons.

CAMP regulates the progesterone receptor gene expression through the protein kinase A pathway during decidualization in human immortalized endometrial stromal cells

Decidualization, a crucial process for successful pregnancy establishment and maintenance, involves endometrial stromal cell differentiation. This process is orchestrated by estradiol (E2), progesterone, and other stimuli that increase intracellular cyclic adenosine monophosphate (cAMP) levels. The intracellular progesterone receptor (PR), encoded by the PGR gene, has a key role in decidualization.This study aimed to understand the role of sex steroids and cAMP in regulating PGR expression during the in vitro decidualization of the human immortalized endometrial stromal cell line, T-HESC. We subjected the cells to individual and combined treatments of E2, medroxyprogesterone (MPA), and cAMP. Additionally, we treated cells with PR and estrogen receptor antagonists and a protein kinase A (PKA) inhibitor. We evaluated the expression of PGR isoforms and decidualization-associated genes by RT-qPCR.Our findings revealed that cAMP induced PGR-B and PGR-AB expression by activating the PKA signaling pathway, while MPA downregulated their expression through the PR. Furthermore, downstream genes involved in decidualization, such as those coding for prolactin (PRL), insulin-like growth factor-binding protein-1 (IGFBP1), and Dickkopf-1 (DKK1), exhibited positive regulation via the cAMP-PKA pathway. Remarkably, MPA-activated PR signaling induced the expression of IGFBP1 and DKK1 but inhibited that of PRL.In conclusion, we have demonstrated that the PKA signaling pathway induces PGR gene expression during in vitro decidualization of the T-HESC human endometrial stromal cell line. This study has unraveled some of the intricate regulatory mechanisms governing PGR expression during this fundamental process for implantation and pregnancy maintenance.

AKAP3-mediated type I PKA signaling is required for mouse sperm hyperactivation and fertility†.

The protein kinase A (PKA) signaling pathway, which mediates protein phosphorylation, is important for sperm motility and male fertility. This process relies on A-kinase anchoring proteins that organize PKA and its signalosomes within specific subcellular compartments. Previously, it was found that the absence of A-kinase anchoring protein 3 (AKAP3) leads to multiple morphological abnormalities in mouse sperm. But how AKAP3 regulates sperm motility is yet to be elucidated. AKAP3 has two amphipathic domains, here named dual and RI, in its N-terminus. These domains are responsible for binding regulatory subunits I alpha (RIα) and II alpha (RIIα) of PKA and for RIα only, respectively. Here, we generated mutant mice lacking the dual and RI domains of AKAP3. It was found that the deletion of these domains caused male mouse infertile, accompanied by mild defects in the fibrous sheath of sperm tails. Additionally, the levels of serine/threonine phosphorylation of PKA substrates and tyrosine phosphorylation decreased in the mutant sperm, which exhibited a defect in hyperactivation under capacitation conditions. The protein levels of PKA subunits remained unchanged. But, interestingly, the regulatory subunit RIα was mis-localized from principal piece to midpiece of sperm tail, whereas this was not observed for RIIα. Further protein-protein interaction assays revealed a preference for AKAP3 to bind RIα over RIIα. Collectively, our findings suggest that AKAP3 is important for sperm hyperactivity by regulating type-I PKA signaling pathway mediated protein phosphorylation via its dual and RI domains.

The protein kinase A signaling pathway mediates the effect of electroacupuncture on excessive contraction of the bladder detrusor in a rat model of neurogenic bladder.

Neurogenic bladder (NB) is a form of neurological bladder dysfunction characterized by excessive contraction of the bladder detrusor. Protein kinase A (PKA) signaling is involved in the contraction of the detrusor muscle. To investigate whether PKA signaling mediates the effect of electroacupuncture (EA) on the excessive contraction of the bladder detrusor in NB. Sixty rats were randomly divided into control, sham, NB, NB + EA, and NB + EA + H89 (a PKA receptor antagonist) groups. The modified Hassan Shaker spinal cord transection method was used to generate a NB model. After EA intervention for one week, urodynamic tests were used to evaluate bladder function, hematoxylin and eosin staining was conducted to assess morphological changes, enzyme-linked immunosorbent assay (ELISA) was performed to measure the concentration of PKA, and Western blotting was conducted to measure the protein levels of phosphorylated myosin light chain kinase (p-MLCK)/p-MLC. The results showed that NB resulted in morphological disruption, impairment of urodynamics, and decreases in the concentration of PKA and the protein levels of p-MLCK/p-MLC. EA reversed the changes induced by NB dysfunction. However, the improvement in urodynamics and the increases in the concentration of PKA and the protein levels of p-MLCK/p-MLC were inhibited by H89. Our findings indicate that the PKA signaling pathway mediates the beneficial effect of EA on excessive contraction of the bladder detrusor in a rat model of NB.

CCK-8 enhances acid-sensing ion channel currents in rat primary sensory neurons

Cholecystokinin (CCK) is a peptide that has been implicated in pain modulation. Acid sensitive ion channels (ASICs) also play an important role in pain associated with tissue acidification. However, it is still unclear whether there is an interaction between CCK signaling and ASICs during pain process. Herein, we report that a functional link between them in rat dorsal root ganglion (DRG) neurons. Pretreatment with CCK-8 concentration-dependently increased acid-evoked ASIC currents. CCK-8 increased the maximum response of ASICs to acid, but did not changed their acid sensitivity. Enhancement of ASIC currents by CCK-8 was mediated by the stimulation of CCK2 receptor (CCK2R), rather than CCK1R. The enhancement of ASIC currents by CCK-8 was prevented by application of either G-protein inhibitor GDP-β-S or protein kinase C (PKC) inhibitor GF109203×, but not by protein kinase A (PKA) inhibitor H-89 or JNK inhibitor SP600125. Moreover, CCK-8 increased the number of action potentials triggered by acid stimuli by activating CCK2R. Finally, CCK-8 dose-dependently exacerbated acid-induced nociceptive behavior in rats through local CCK2R. Together, these results indicated that CCK-8/CCK2R activation enhanced ASIC-mediated electrophysiological activity in DRG neurons and nociception in rats. The enhancement effect depended on G-proteins and intracellular PKC signaling rather than PKA and JNK signaling pathway. These findings provided that CCK-8/CCK2R is an important therapeutic target for ASIC-mediated pain.

Oleanolic acid alleviates the extrapyramidal symptoms and cognitive impairment induced by haloperidol through the striatal PKA signaling pathway in mice

Haloperidol, one of the representative typical antipsychotics, is on the market for schizophrenia but shows severe adverse effects such as extrapyramidal symptoms (EPS) or cognitive impairments. Oleanolic acid (OA) is known to be effective for tardive dyskinesia which is induced by long-term treatment with L-DOPA. This study aimed to investigate whether OA could ameliorate EPS or cognitive impairment induced by haloperidol. The balance beam, catalepsy response, rotarod and vacuous chewing movement (VCM) tests were performed to measure EPS and the novel object recognition test was used to estimate haloperidol-induced cognitive impairment. Levels of dopamine and acetylcholine, the phosphorylation levels of c-AMP-dependent protein kinase A (PKA) and its downstream signaling molecules were measured in the striatum. OA significantly attenuated EPS and cognitive impairment induced by haloperidol without affecting its antipsychotic properties. Valbenazine only ameliorated VCM. Also, OA normalised the levels of dopamine and acetylcholine in the striatum which were increased by haloperidol. Furthermore, the increased phosphorylated PKA, extracellular signal-regulated kinase (ERK) and cAMP response element-binding protein (CREB) levels and c-FOS expression level induced by haloperidol were significantly decreased by OA in the striatum. In addition, cataleptic behaviour of haloperidol was reversed by sub-effective dose of H-89 with OA. These results suggest that OA can alleviate EPS and cognitive impairment induced by antipsychotics without interfering with antipsychotic properties via regulating neurotransmitter levels and the PKA signaling pathway in the striatum. Therefore, OA is a potential candidate for treating EPS and cognitive impairment induced by antipsychotics.

Etomidate Depresses Spontaneous Complex Spikes Activity of Cerebellar Purkinje Cells via Cannabinoid 1 Receptor in vivo in Mice

Introduction: Complex spikes (CSs) activity of cerebellar Purkinje cells plays critical roles in motor coordination and motor learning by transferring information to cerebellar cortex, which is an accessible and useful model for neurophysiological investigation. Etomidate is an ultrashort-acting nonbarbiturate intravenous anesthetic, which inhibits the spontaneous activity of cerebellar Purkinje cells through activation of GABA_A and glycine receptors in vivo in mice. However, the effect of etomidate on the spontaneous CSs activity of cerebellar Purkinje cells in living mouse is not clear. Methods: We here investigated the effects of etomidate on spontaneous CSs activity of cerebellar Purkinje cell in urethane-anesthetized mice by electrophysiology recording technique and pharmacological methods. Results: Our results showed that cerebellar surface perfusion of etomidate significantly depressed the activity of spontaneous CSs, which exhibited decreases in the number of spikelets and the area under curve (AUC) of the CSs. The etomidate-produced inhibition of CSs activity was persisted in the presence of GABA_A and glycine receptors antagonists. However, application of cannabinoid 1 (CB1) receptor antagonist, AM-251, completely blocked the etomidate-induced inhibition of CSs. Furthermore, application of the CB1 receptor agonist, WIN55212-2, induced a decrease of CSs. Moreover, in the presence of a specific protein kinase A (PKA) inhibitor, KT5720, etomidate failed to produce decreases in the spikelets number and the AUC of the spontaneous CSs. Conclusion: These results indicate that cerebellar surface application of etomidate facilitates CB1 receptor activity resulting in a depression of spontaneous CSs activity of Purkinje cells via PKA signaling pathway in mouse cerebellar cortex. Our present results suggest that the etomidate administration may impair the function of cerebellar cortical neuronal circuitry by inhibition of the climbing fiber – Purkinje cells synaptic transmission through activation of CB1 receptors in vivo in mice.

The role of cAMP-dependent protein kinase A in the formation of long-term memory in Bactrocera dorsalis

The cAMP-dependent protein kinase A (PKA) signaling pathway has long been considered critical for long-term memory (LTM) formation. Previous studies have mostly focused on the role of PKA signaling in LTM induction by multiple spaced conditioning with less attention to LTM induction by a single conditioning. Here, we conducted behavioral-pharmacology, enzyme immunoassay and RNA interference experiments to study the role of the PKA signaling pathway in LTM formation in the agricultural pest Bactrocera dorsalis, which has a strong memory capacity allowing it to form a two-day memory even from a single conditioning trial. We found that either blocking or activating PKA prior to conditioning pretreatment affected multiple spaced LTM, and conversely, they did not affect LTM formed by single conditioning. This was further confirmed by enzyme-linked immunosorbent assay (ELISA) and silencing of the protein kinase regulatory subunit 2 and catalytic subunit 1. Taken together, these results suggest that activating PKA during memory acquisition helps to induce the LTM formed by multiple spaced conditioning but not by a single conditioning. Our findings challenge the conserved role of PKA signaling in LTM, which provides a basis for the greater diversity of molecular mechanisms underlying LTM formation across species, as well as possible functional and evolutionary implications.

Vitamin D3 protects against respiratory syncytial virus-induced barrier dysfunction in airway epithelial cells via PKA signaling pathway

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infection in infants and young children globally and is responsible for hospitalization and mortality in the elderly population. Virus-induced airway epithelial barrier damage is a critical step during RSV infection, and emerging studies suggest that RSV disrupts the tight junctions (TJs) and adherens junctions (AJs) between epithelial cells, increasing the permeability of the airway epithelial barrier. The lack of commercially available vaccines and effective antiviral drugs for RSV emphasizes the need for new management strategies. Vitamin D3 is a promising intervention for viral infection due to its critical role in modulating innate immune responses. However, there is limited evidence on the effect of vitamin D3 on RSV pathogenies. Here, we investigated the impact of vitamin D3 on RSV-induced epithelial barrier dysfunction and the underlying mechanisms. We found that pre-incubation with 1,25(OH)2D3, the active form of vitamin D3, alleviated RSV-induced epithelial barrier disruption in a dose-dependent manner without affecting viability in 16HBE cells. 1,25(OH)2D3 induced minor changes in the protein expression level of TJ/AJ proteins in RSV-infected cells. We observed increased CREB phosphorylation at Ser133 during 1,25(OH)2D3 exposure, indicating that vitamin D3 triggered protein kinase A (PKA) activity in 16HBE. PKA inhibitors modified the restoration of barrier function by 1,25(OH)2D3 in RSV-infected cells, implying that PKA signaling is responsible for the protective effects of vitamin D3 against RSV-induced barrier dysfunction in airway epithelial cells. Our findings suggest vitamin D3 as a prophylactic intervention to protect the respiratory epithelium during RSV infections.

Abstract 5: Activation of PKA Signaling: A Milestone Associated With Bone Progression of Prostate Cancer

Abstract Purpose: Metabolic rewiring is critical for the adaptation of tumor cells to the new homing organ. We sought to identify metabolic dysregulations fueling PCa bone metastasis, modulated by bone secreted factors. Methods: By an indirect co-culture system of PCa (PC3) and bone progenitor cells (MC3T3, pre-osteoblasts, or Raw264.7, pre-osteoclasts), we assessed the transcriptome of PC3 cells modulated by soluble factors released by bone precursors. We validated the transcriptional profile of metabolic genes using open-access transcriptomic datasets. We performed an Ingenuity Pathway Analysis (IPA) to delineate the regulators of these metabolic genes. The bone secretome was profiled in the conditioned media (CM) by ESI-MS/MS. We validated our results using a PDX pre-clinical model comparing gene expression levels in MDA-PCa-183 growing intrafemorally (i.f.) vs. subcutaneously (s.c.). Results: PC3 cells co-cultured with bone progenitors displayed an activation of lipid metabolism, including the PPAR-signaling and fat absorption/digestion pathways. Accordingly, we observed an accumulation of neutral lipids in PC3 cells treated with the CM from the co-culture (Bodipy 493/503 staining). Unsupervised Clustering analysis using transcriptomic data from human PCa and bone metastatic samples (GSE74685) showed that the metabolic genes deregulated in co-cultured PC3 accurately clustered samples in primary tumor or bone metastasis. Moreover, 5 lipid-associated genes, PPARA, VDR, SLC16A1, PAPSS2 and GPX1, were associated with a 23-fold higher risk of death (SU2C-PCF dataset). This expression signature was validated in a PDX pre-clinical model when comparing MDA-PCa-183 growing i.f. vs. s.c. An IPA revealed that these genes are regulated by the Protein Kinase A (PKA). Accordingly, PKA inhibition led to a downregulation of these genes. Moreover, GSEA showcased an activation of PKA signaling pathways in MDA-PCa-183 i.f. vs. s.c., reinforcing the fact that the bone environment shapes PCa metabolism. Additionally, secretome and protein-protein interaction analyses revealed soluble factors (Col1a1, Fn1, etc.) secreted by bone cells that could regulate PKA activity. Conclusion: We identified a novel lipid-associated gene signature important for metastatic PCa, triggered by the dialogue with bone cells. This signature is under the regulation of PKA in response to bone-secreted factors, emerging as a potential target for intervention. Citation Format: Pablo Sanchis, Nicolas Anselmino, Sofia Lage-Vickers, Agustina Sabater, Rosario Lavignolle, Estefania Labanca, Peter Shepherd, Juan Bizzotto, Gaston Pascual, Rocio Seniuk, Ayelen Toro, Antonina Mitrofanova, Pia Valacco, Nora Navone, Javier Cotignola, Elba Vazquez, Geraldine Gueron. Activation of PKA Signaling: A Milestone Associated With Bone Progression of Prostate Cancer [abstract]. In: Proceedings of the 11th Annual Symposium on Global Cancer Research; Closing the Research-to-Implementation Gap; 2023 Apr 4-6. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(6_Suppl):Abstract nr 5.

Role of the protein kinase A signaling pathway and identification of mediators in the cardioprotective effects of enteral lactoferrin for ischemia-reperfusion injury in an isolated rat heart model

ObjectiveLactoferrin is an iron-binding glycoprotein. Enteral lactoferrin attenuates myocardial ischemia-reperfusion (IR) injury, but the underlying mechanism remains unknown. The aim of this study was to investigate protein kinase A (PKA) signaling pathway activation and levels of serum glucagonlike peptide-1 (GLP-1), secreted by intestinal endocrine L cells, and adiponectin, secreted by adipose tissue, after enteral lactoferrin administration. MethodsHearts (N = 32) were excised from Wistar rats and perfused using a Langendorff system. To assess the role of the PKA pathway in the cardioprotective effects of lactoferrin, an inhibitor of PKA (H89) was applied before no-flow ischemia. Rats were randomly divided into four groups: control, lactoferrin (LF), control+H89, and LF+H89. The control and control+H89 groups were administered normal saline by gavage, and the LF and L +H89 groups were administered bovine lactoferrin (1000 mg/kg) by gavage 15 min before intraperitoneal pentobarbital injection. Muscle sampling was performed at the end of reperfusion. When rats were sacrificed, blood was sampled to measure hormone levels. The primary outcome was maximum left ventricular pressure derivative (LV dP/dt max) 15 min after reperfusion. ResultsLV dP/dt max at 10 and 15 min after reperfusion was significantly higher in the LF group than in the control group (P < 0.05), and the effect was diminished by H89. The PKA pathway was significantly activated in the LF group. Enteral lactoferrin increased serum GLP-1 but not serum adiponectin levels. ConclusionsEnteral lactoferrin induces cardioprotective effects against myocardial IR injury via the PKA signaling pathway and increases serum GLP-1 levels.

Glucagon-like peptide-1 facilitates cerebellar parallel fiber glutamate release through PKA signaling in mice in vitro

Glucagon-like peptide-1 (GLP-1) is mainly secreted by preproglucagon neurons; it plays important roles in modulating neuronal activity and synaptic transmission through its receptors. In the present study, we investigated the effects of GLP-1 on parallel fiber–Purkinje cell (PF-PC) synaptic transmission in mouse cerebellar slices using whole-cell patch-clamp recording and pharmacology methods. In the presence of a γ-aminobutyric acid type A receptor antagonist, bath application of GLP-1 (100 nM) enhanced PF-PC synaptic transmission, with an increased amplitude of evoked excitatory postsynaptic synaptic currents (EPSCs) and a decreased paired-pulse ratio. The GLP-1-induced enhancement of evoked EPSCs was abolished by a selective GLP-1 receptor antagonist, exendin 9–39, as well as by the extracellular application of a specific protein kinase A (PKA) inhibitor, KT5720. In contrast, inhibiting postsynaptic PKA with a protein kinase inhibitor peptide-containing internal solution failed to block the GLP-1-induced enhancement of evoked EPSCs. In the presence of a mixture of gabazine (20 μM) and tetrodotoxin (1 μM), application GLP-1 significantly increased frequency, but not amplitude of miniature EPSCs via PKA signaling pathway. The GLP-1-induced increase in miniature EPSC frequency was blocked by both exendin 9–39 and KT5720. Together, our results indicate that GLP-1 receptor activation enhances glutamate release at PF-PC synapses via the PKA signaling pathway, resulting in enhanced PF-PC synaptic transmission in mice in vitro. These findings suggest that, in living animals, GLP-1 has a critical role in the modulation of cerebellar function by regulating excitatory synaptic transmission at PF-PC synapses.

Regulation of Transcriptional Activity of Merkel Cell Polyomavirus Large T-Antigen by PKA-Mediated Phosphorylation.

Merkel cell polyomavirus (MCPyV) is the major cause of Merkel cell carcinoma (MCC), an aggressive skin cancer. MCPyV large T-antigen (LTag) and small T-antigen (sTag) are the main oncoproteins involved in MCPyV-induced MCC. A hallmark of MCPyV-positive MCC cells is the expression of a C-terminal truncated LTag. Protein kinase A (PKA) plays a fundamental role in a variety of biological processes, including transcription by phosphorylating and thereby regulating the activity of transcription factors. As MCPyV LTag has been shown to be phosphorylated and acts as a transcription factor for the viral early and late promoter, we investigated whether LTag can be phosphorylayted by PKA, and whether this affects the transcript activity of LTag. Using a phosphorylation prediction algorithm, serine 191, 203, and 265 were identified as putative phosphorylation sites for PKA. Mass spectrometry of in vitro PKA-phosphorylated peptides confirmed phosphorylation of S203 and S265, but not S191. Full-length LTag inhibited early and late promoter activity of MCPyV, whereas the truncated MKL2 LTag variant stimulated both promoters. Single non-phosphorylable, as well as phosphomimicking mutations did not alter the inhibitory effect of full-length LTag. However, the non-phosphorylable mutations abrogated transactivation of the MCPyV promoters by MKL2 LTag, whereas phosphomimicking substitutions restored the ability of MKL2 LTag to activate the promoters. Triple LTag and MKL2 LTag mutants had the same effect as the single mutants. Activation of the PKA signaling pathway did not enhance MCPyV promoter activity, nor did it affect LTag expression levels in MCPyV-positive Merkel cell carcinoma (MCC) cells. Our results show that phosphorylation of truncated LTag stimulates viral promoter activity, which may contribute to higher levels of the viral oncoproteins LTag and sTag. Interfering with PKA-induced LTag phosphorylation/activity may be a therapeutic strategy to treat MCPyV-positive MCC patients.

Neurotensin expression, regulation, and function during the ovulatory period in the mouse ovary†.

The luteinizing hormone (LH) surge induces paracrine mediators within the ovarian follicle that promote ovulation. The present study explores neurotensin (NTS), a neuropeptide, as a potential ovulatory mediator in the mouse ovary. Ovaries and granulosa cells (GCs) were collected from immature 23-day-old pregnant mare serum gonadotropin primed mice before (0h) and after administration of human chorionic gonadotropin (hCG; an LH analog) across the periovulatory period (4, 8, 12, and 24h). In response to hCG, Nts expression rapidly increased 250-fold at 4h, remained elevated until 8h, and decreased until 24h. Expression of Nts receptors for Ntsr1 remained unchanged across the periovulatory period, Ntsr2 was undetectable, whereas Sort1 expression (also called Ntsr3) gradually decreased in both the ovary and GCs after hCG administration. To better understand Nts regulation, inhibitors of the LH/CG signaling pathways were utilized. Our data revealed that hCG regulated Nts expression through the protein kinase A (PKA) and p38 mitogen-activated protein kinase (p38MAPK) signaling pathways. Additionally, epidermal-like-growth factor (EGF) receptor signaling also mediated Nts induction in GCs. To elucidate the role of NTS in the ovulatory process, we used a Nts silencing approach (si-Nts) followed by RNA-sequencing (RNA-seq). RNA-seq analysis of GCs collected after hCG with or without si-Nts identified and qPCR confirmed Ell2, Rsad2, Vps37a, and Smtnl2 as genes downstream of Nts. In summary, these findings demonstrate that hCG induces Nts and that Nts expression is mediated by PKA, p38MAPK, and EGF receptor signaling pathways. Additionally, NTS regulates several novel genes that could potentially impact the ovulatory process.

The antidiabetic drug teneligliptin induces vasodilation via activation of PKG, Kv channels, and SERCA pumps in aortic smooth muscle

Diabetes mellitus (DM) is a metabolic disease closely related to cardiovascular disease. The dipeptidyl peptidase-4 inhibitor teneligliptin is used to treat DM and has recently been shown to have a cardiovascular protective effect against diseases such as hypertension and heart failure. The present study demonstrates the vasodilatory effect of teneligliptin using aortic rings pre-contracted with phenylephrine. Teneligliptin induced a vasodilatory effect in a dose-dependent manner, with and without endothelium. In addition, pretreatment with the nitric oxide synthase inhibitor L-NAME and small-conductance Ca2+-activated K+ channel inhibitor apamin did not alter the teneligliptin-induced vasodilatory effect. Although the adenylyl cyclase inhibitor SQ 22536 and protein kinase A (PKA) inhibitor KT 5720 did not modulate the vasodilatory effect of teneligliptin, the guanylyl cyclase inhibitor ODQ and protein kinase G (PKG) inhibitor KT 5823 effectively reduced the effect of teneligliptin. Similarly, pretreatment with the voltage-dependent K+ (Kv) channel inhibitor 4-aminopyridine (4-AP) also reduced teneligliptin-induced vasodilation. However, pretreatment with the inward rectifier K+ (Kir) channel inhibitor Ba2+, large-conductance Ca2+-activated K+ (BKCa) channel inhibitor paxilline, and ATP-sensitive K+ (KATP) channel inhibitor glibenclamide did not alter the vasodilatory effect of teneligliptin. Our data suggest that Kv7.X, but not Kv1.5 or Kv2.1, is one of the major Kv subtypes involved in teneligliptin-induced vasodilation. Furthermore, pretreatment with the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitor thapsigargin and CPA inhibited the vasodilation induced by teneligliptin. Our results suggest that teneligliptin-induced vasodilation occurs via activation of PKG, SERCA pumps and Kv channels, but not the PKA signaling pathway, other K+ channels, or endothelium.

Impact of synemin protein, an intermediate filament, in cardiac physiopathology

Synemin is a protein belonging to the intermediate filament family involved in the cytoarchitecture of skeletal, cardiac and smooth muscle cells. Synemin copolymerizes with other intermediate filaments (desmin, vimentin) and interacts with sarcomeric and costameric proteins, acting as bridging protein. The tail domain of synemin ties the type II α regulatory (R) subunit of protein kinase A (PKA). Therefore, synemin is an A-kinase-anchoring protein (AKAP) that allows and modulates spatial and temporal targeting of its activity. We have previously found that mechanical overload of skeletal muscle induces greater hypertrophy of muscle fibres in synemin-knockout mice associated with an impairment of PKA signalling pathways. In humans, two missense mutations, located near the AKAP domain, have been recorded in patients with dilated cardiomyopathy. The aims are: (1) To analyze the role of synemin in cardiac hypertrophy; (2) To characterize the signalling pathways related to synemin in the hypertrophic response. Morphological (body and heart weights), cellular (immunostaining) and molecular (RT-qPCR, Western blot) analyzes were carried out on cardiomyocyte-specific synemin-knockout mice at 10, 30 and 60 days after tamoxifen injections. Then, we studied the response of the hypertrophic agent angiotensin II (Ang II) after 14 days delivered by Alzet osmotic mini-pumps in these mice. Our data show a decrease (> 50%) of synemin expression in heart of conditional knockout mice 10 days after tamoxifen injections. Thus, we observed an increase of stress gene expression (ANF, βMHC) and fibrotic markers (vimentin, coll3α1). Immunofluorescence of vimentin on heart sections reveals a strong increase of protein expression suggesting an activation of fibroblast proliferation. Chronic administration of Ang II does not show modification in gene expression profile between control and conditional synemin-knockout mice. At 30 and 60 days after tamoxifen injections, we observed the maintenance of mRNA synemin level indicating a compensatory expression of this protein by other cells in heart (fibroblasts, macrophages, endothelial and smooth cells). Our data indicate a crucial role of synemin as modulator in the physiopathological conditions. Moreover, the relevance of synemin-PKA coupling in cardiac hypertrophy will be characterized in the near future.

Genetic Alterations in Benign Adrenal Tumors

The genetic basis of most types of adrenal adenomas has been elucidated over the past decade, leading to the association of adrenal gland pathologies with specific molecular defects. Various genetic studies have established links between variants affecting the protein kinase A (PKA) signaling pathway and benign cortisol-producing adrenal lesions. Specifically, genetic alterations in GNAS, PRKAR1A, PRKACA, PRKACB, PDE11A, and PDE8B have been identified. The PKA signaling pathway was initially implicated in the pathogenesis of Cushing syndrome in studies aiming to understand the underlying genetic defects of the rare tumor predisposition syndromes, Carney complex, and McCune-Albright syndrome, both affected by the same pathway. In addition, germline variants in ARMC5 have been identified as a cause of primary bilateral macronodular adrenal hyperplasia. On the other hand, primary aldosteronism can be subclassified into aldosterone-producing adenomas and bilateral idiopathic hyperaldosteronism. Various genes have been reported as causative for benign aldosterone-producing adrenal lesions, including KCNJ5, CACNA1D, CACNA1H, CLCN2, ATP1A1, and ATP2B3. The majority of them encode ion channels or pumps, and genetic alterations lead to ion transport impairment and cell membrane depolarization which further increase aldosterone synthase transcription and aldosterone overproduction though activation of voltage-gated calcium channels and intracellular calcium signaling. In this work, we provide an overview of the genetic causes of benign adrenal tumors.

Silibinin promotes melanogenesis through the PKA and p38 MAPK signaling pathways in melanoma cells.

Silibinin is a flavonolignan isolated from milk thistle (Silybum marianum). Silibinin has been reported to possess multiple biological activities; however, its effect on melanogenesis remains unclear. This study investigated the effect of silibinin on melanogenesis in melanoma cells and the associated molecular mechanism. Our findings demonstrated that silibinin markedly increased melanin content in murine B16-F1 and human HMV-II melanoma cells. Silibinin activated intracellular tyrosinase activity and expression of tyrosinase, tyrosinase-related protein (TRP)-1, TRP-2, and microphthalmia-associated transcription factor (MITF). Furthermore, silibinin enhanced the phosphorylation of cyclic AMP-responsive element-binding protein (CREB), protein kinase A (PKA), and p38 mitogen-activated protein kinase (MAPK) but not of Akt and extracellular signal-regulated kinase (ERK). The specific PKA (H-89) and p38 (SB203580) inhibitors significantly attenuated silibinin-mediated melanin synthesis. These results suggest that silibinin is an effective stimulator of melanogenesis through upregulation of the protein expression of melanogenic enzymes activated by the PKA and p38 pathways, leading to CREB phosphorylation and MITF expression. Therefore, silibinin may have potential for use in the treatment of hypopigmentation disorders.