Protein Kinase A Activity Research Articles

Novel insights in the pathomechanism of Brugada syndrome and fever-related type 1 ECG changes in a preclinical study using human-induced pluripotent stem cell-derived cardiomyocytes.

Brugada syndrome (BrS) is causing sudden cardiac death (SCD) mainly at young age. Studying the underlying mechanisms associated with BrS type I electrocardiogram (ECG) changes in the presence of fever and roles of autophagy for BrS remains lacking. We sought to study the pathogenic role of an SCN5A gene variant for BrS with fever-induced type 1 ECG phenotype. In addition, we studied the role of inflammation and autophagy in the pathomechanism of BrS. Human-induced pluripotent stem cell (hiPSC) lines from a BrS patient harboring a pathogenic variant (c.3148G>A/p. Ala1050Thr) in SCN5A and two healthy donors (non-BrS) and a CRISPR/Cas9 site-corrected cell line (BrS-corr) were differentiated into cardiomyocytes (hiPSC-CMs) for the study. Reductions of Nav 1.5 expression, peak sodium channel current (INa ) and upstroke velocity (Vmax ) of action potentials with an increase in arrhythmic events were detected in BrS compared to non-BrS and BrS-corr cells. Increasing the cell culture temperature from 37 to 40°C (fever-like state) exacerbated the phenotypic changes in BrS cells. The fever-effects were enhanced by protein kinase A (PKA) inhibitor but reversed by PKA activator. Lipopolysaccharides (LPS) but not increased temperature up to 40°C enhanced the autophagy level in BrS-hiPSC-CMs by increasing reactive oxidative species and inhibiting PI3K/AKT signalling, and hence exacerbated the phenotypic changes. LPS enhanced high temperature-related effect on peak INa shown in BrS hiPSC-CMs. Effects of LPS and high temperature were not detected in non-BrS cells. The study demonstrated that the SCN5A variant (c.3148G>A/p.Ala1050Thr) caused loss-of-function of sodium channels and increased the channel sensitivity to high temperature and LPS challenge in hiPSC-CMs from a BrS cell line with this variant but not in two non-BrS hiPSC-CM lines. The results suggest that LPS may exacerbate BrS phenotype via enhancing autophagy, whereas fever may exacerbate BrS phenotype via inhibiting PKA-signalling in BrS cardiomyocytes with but probably not limited to this variant.

MiR-34c-3p Regulates Protein Kinase A Activity Independent of cAMP by Dicing prkar2b Transcripts in Theileria annulata-Infected Leukocytes.

MicroRNAs (miRNAs) are small noncoding RNAs that can play critical roles in regulating various cellular processes, including during many parasitic infections. Here, we report a regulatory role for miR-34c-3p in cAMP-independent regulation of host cell protein kinase A (PKA) activity in Theileria annulata-infected bovine leukocytes. We identified prkar2b (cAMP-dependent protein kinase A type II-beta regulatory subunit) as a novel miR-34c-3p target gene and demonstrate how infection-induced upregulation of miR-34c-3p repressed PRKAR2B expression to increase PKA activity. As a result, the disseminating tumorlike phenotype of T. annulata-transformed macrophages is enhanced. Finally, we extend our observations to Plasmodium falciparum-parasitized red blood cells, where infection-induced augmentation in miR-34c-3p levels led to a drop in the amount of prkar2b mRNA and increased PKA activity. Collectively, our findings represent a novel cAMP-independent way of regulating host cell PKA activity in infections by Theileria and Plasmodium parasites. IMPORTANCE Small microRNA levels are altered in many diseases, including those caused by parasites. Here, we describe how infection by two important animal and human parasites, Theileria annulata and Plasmodium falciparum, induce changes in infected host cell miR-34c-3p levels to regulate host cell PKA kinase activity by targeting mammalian prkar2b. Infection-induced changes in miR-34c-3p levels provide a novel epigenetic mechanism for regulating host cell PKA activity independent of fluxes in cAMP to both aggravate tumor dissemination and improve parasite fitness.

Role of Protein Kinase A Activation in the Immune System with an Emphasis on Lipopolysaccharide-Responsive and Beige-like Anchor Protein in B Cells

Cyclic AMP-dependent protein kinase A (PKA) is a ubiquitous enzymatic complex that is involved in a broad spectrum of intracellular receptor signaling. The activity of PKA depends on A-kinase anchoring proteins (AKAPs) that attach to PKAs close to their substrates to control signaling. Although the relevance of PKA-AKAP signaling in the immune system is evident in T cells, its relevance in B and other immune cells remains relatively unclear. In the last decade, lipopolysaccharide-responsive and beige-like anchor protein (LRBA) has emerged as an AKAP that is ubiquitously expressed in B and T cells, specifically after activation. A deficiency of LRBA leads to immune dysregulation and immunodeficiency. The cellular mechanisms regulated by LRBA have not yet been investigated. Therefore, this review summarizes the functions of PKA in immunity and provides the most recent information regarding LRBA deficiency to deepen our understanding of immune regulation and immunological diseases.

Protein kinase A activation alleviates cataract formation via increased gap junction intercellular communication

Cataract is the leading cause of blindness worldwide. Here, we reported a potential, effective therapeutic mean for cataract prevention and treatment. Gap junction communication, an important mechanism in maintaining lens transparency, is increased by protein kinase A (PKA). We found that PKA activation reduced cataracts induced by oxidative stress, increased gap junctions/hemichannels in connexin (Cx) 50, Cx46 or Cx50 and Cx46 co-expressing cells, and decreased reactive oxygen species (ROS) levels. However, ROS reduction was shown in wild-type, Cx46 and Cx50 knockout, but not in Cx46/Cx50 double KO lens. In addition, PKA activation protects lens fiber cell death induced by oxidative stress via hemichannel-mediated glutathione transport. Connexin deletion increased lens opacity induced by oxidative stress associated with reduction of anti-oxidative stress gene expression. Together, our results suggest that PKA activation through increased connexin channels in lens fiber cell decreases ROS levels and cell death, leading to alleviated cataracts.

Protein kinase A (PKA) activation reorganizes microtubules and microtubule-plus-end-binding-protein 1 (EB1) and enriches Nav1.5 at the intercalated discs (ICDs) of adult ventricular myocytes.

In cardiomyocytes β-adrenergic stimulation increases Na current (INa) through PKA activation, and EB1 targets Nav1.5 to ICDs where INa mediates impulse propagation through gap-junctions. To understand how persistent PKA activation affects INa and Nav1.5 protein expression/distribution in cardiomyocytes, and the role of EB1 and microtubules. Sprague-Dawley ventricular myocytes cultured without or with 8cpt-cAMP (100 uM) plus serine/threonine phosphatase inhibitor (okadaic acid, 100 nM) for 10-20 hr were subject to immunostaining/imaging, subcellular fractionation/immunoblot, and patch clamp recordings. These were supplemented by HEK293 experiments. Cell-attached patch recordings from ICD areas (≤5 sarcomeres from cell ends) showed 10-fold increase in INa in PKA-activated vs control myocytes, without change in total Nav1.5 protein level. To understand Nav1.5 distribution and relationship to EB1 and microtubules at ICDs, we imaged the 3 with n-cadherin (ICD marker) simultaneously using Airyscan at xyz pixel dimension 50 nm. Image reconstruction allowed us to quantify protein signals in en-face views of ICDs. PKA activation (a) increased %ICD area occupied by Nav1.5, β-tubulin and EB1, and (b) increased correlation between Nav1.5/EB1, Nav1.5/β-tubulin, and EB1/β-tubulin. These were supported by HEK293 data: Nav1.5 co-immunoprecipitated more EB1 and β-tubulin in PKA activated (4-6 hr) than control cells. Although PKA activation increased EB1 capture of microtubules at ICD, it decreased microtubule density in the cytoplasmic area, consistent with biochemical data: PKA activation did not change total α-tubulin level but reduced detyrosinated α-tubulin. Myocytes have >10 fold free β-tubulin in cytosol than in microtubules, and >150 fold free EB1 in cytosol than bound to microtubules/membranes. Spatial/temporal dynamics of microtubules and EB1 modulation and PKA-promoted Nav1.5/tubulin/EB1 interactions enrich Nav1.5 at ICDs, facilitating impulse propagation in β-adrenergic stimulated heart.

5-adenosine monophosphate promotes signal adaptation in PKA signaling.

Protein Kinase A (PKA) plays a central role in several cellular processes and its activity is tightly regulated by 2nd messenger 3,5-cyclic adenosine monophosphate (cAMP)-signaling. While the role of cAMP in PKA activation is well established, the significance of its hydrolysis end-product, 5-adenosine monophosphate (AMP) is poorly understood. AMP is generated upon hydrolysis of cAMP bound to the PKA regulatory subunits (RIα) catalyzed by phosphodiesterases. All components of the cAMP-PKA signaling pathway that include PDEs are compartmentalized into signalosomal units. Our research has shown that PDEs in proximity with PKA RIα in signalosomes lead to formation of a signal termination complex of PDEs and RIα, that facilitates cAMP substrate channeling from the PKA binding site to the PDE catalytic site. In contrast to the long held view that AMP diffused away from the PKA signalosome following cAMP hydrolysis, AMP is a regulatory end-product of each round of PKA activation and termination. Here we report high resolution structures of the AMP bound conformations of RIα and PDE8A catalytic domain (PDE8c) by cryo-EM. We have further captured AMP-bound end-state signal termination complex of PDE8c with RIα, by orthogonal electron microscopy screening, amide hydrogen/deuterium exchange mass spectrometry. Our results describe an undiscovered active role of AMP in signal termination and adaptation in PKA signaling.

Chemigenetic kinase biosensors for multiplexed activity sensing and super-resolution imaging.

Genetically encoded fluorescent biosensors have revolutionized the study of dynamic biochemical processes in living cells. They enable to investigate intertwined signaling pathways both under physiological and pathological conditions. For instance, kinase activity is often regulated through tight spatiotemporal control via compartmentation. Specifically, protein kinase A (PKA) is organized into plasma membrane nanodomains or sequestered in cytoplasmic phase separated bodies. Due to the small size of these compartments, little is known about the PKA activity organization and regulation within them, even though this knowledge would be crucial to untangle intricately regulated PKA signaling. To investigate these signaling compartments with the necessary spatial resolution and under more physiological conditions new biosensors with enhanced capabilities need to be developed.

Caveolae-restricted mechano-chemical signal transduction in mouse atrial myocytes.

Atrial fibrillation (AF) is commonly observed in patients with hypertension and is associated with pathologically elevated cardiomyocyte stretch. AF triggers have been linked to subcellular Ca2+ abnormalities, while their association with stretch remains elusive. Caveolae are mechanosensitive membrane structures, that play a role in both Ca2+ and cyclic adenosine monophosphate (cAMP) signaling. Therefore, caveolae could provide a mechanistic connection between cardiomyocyte stretch, Ca2+ mishandling, and AF. In isolated mouse atrial myocytes, cell stretch was mimicked by hypotonic swelling, which increased cell width (by ∼30%, p<0.05), but not cell length. Swelling induced a temporal rise (1-5 min) in Ca2+ transient (CaT) amplitude and spontaneous Ca2+ sparks frequency (CaSpF). Electron microscopy analysis revealed a 2-fold decrease in caveolae density in stretched cells. Alternatively, disruption of caveolae via (1) 10 mM methyl-β-cyclodextrin (MβCD) or (2) genetic knockout of caveolin-3 augmented both CaT and CaSpF, similar to swelling. Immunofluorescent analysis revealed the augmentation of protein kinase A (PKA)-phosphorylated ryanodine receptors (at both Ser2030 and Ser2808) in swelled and MβCD-treated cells as compared to isotonic ones (p<0.05). CaSpF in swelled and MβCD-treated cells was reduced to the unstretched level via (1) inhibition of cAMP production by 100 μM SQ22536, or (2) reduction of PKA activity by 1 μM H-89. Transfection of myocytes with either sarcolemma- or cytosol-targeted Epac2-camps FRET sensors revealed a sharp decrease of cAMP signal in the submembrane compartment and a transient rise in the cytosol during the stretch. Atrial myocytes from pressure-overloaded hearts (4-weeks transaortic constriction) showed 1.6 times higher CaSpF than wild-type cells (p<0.01), which was significantly reduced after incubation with SQ22536 (p<0.05). Our findings demonstrate that short-time cell swelling augments Ca2+ handling in atrial myocytes via caveolae/cAMP/PKA mechano-chemical signal transduction, which could contribute to atrial arrhythmogenesis under pathological conditions.

Mirror image pain mediated by D2 receptor regulation of astrocytic Cx43 phosphorylation and channel opening

Mirror image pain (MIP), a clinical syndrome of contralateral pain hypersensitivity caused by unilateral injury, has been identified in various neuropathological conditions. Gap junctional protein Connexin 43 (Cx43), its phosphorylation levels and dopamine D2 receptor (DRD2) play key integrating roles in pain processing. We presume D2DR activity may affect Cx43 hemichannel opening via Cx43 phosphorylation levels to regulate MIP. This study shows that spinal astrocytic Cx43 directly interacts with DRD2 to mediate MIP. DRD2 and Cx43 expression levels were asymmetrically elevated in bilateral spinal during MIP, and DRD2 modulated the opening of primary astrocytic Cx43 hemichannels. Furthermore, Cx43 phosphorylation at Ser373 was increased during MIP, but decreased in DRD2 knockout (KO) mice. Finally, activation of spinal protein kinase A (PKA) altered the expression of Cx43 and its phosphorylation bilaterally, thus reversing the analgesic effect in DRD2 KO mice. Together, these data reveal that spinal Cx43 phosphorylation and channel opening are regulated by DRD2 via PKA activation, and that spinal Cx43 and DRD2 are key molecular sensors mediating mirror image pain.

Intranasal Administration of Forskolin and Noopept Reverses Parkinsonian Pathology in PINK1 Knockout Rats.

Parkinson's Disease (PD) is a brain-degenerative disorder characterized by a progressive loss of midbrain dopamine neurons. Current standard-of-care includes oral administration of Levodopa to address motor symptoms, but this treatment is not disease-modifying. A reduction in Protein Kinase A (PKA) signaling and neurotrophic support contributes to PD pathology. We previously showed that enhancing PKA activity in the brain via intraperitoneal administration of Forskolin in Parkinsonian rats (PINK1 knockout) abrogate motor symptoms and loss of midbrain dopamine neurons. Given that intraperitoneal administration is invasive, we hypothesized that intranasal administration of Forskolin and a second nootropic agent (Noopept) could reverse PD pathology efficiently. Results show that intranasal administration of a formulation (CNS/CT-001) containing Forskolin (10 µM) and Noopept (20 nM) significantly reversed motor symptoms, loss of hind limb strength, and neurodegeneration of midbrain dopamine neurons in PINK1-KO rats and is indistinguishable from wild-type (WT) rats; therapeutic effects associated with increased PKA activity and levels of BDNF and NGF in the brain. Intranasal administration of CNS/CT-001, but not Forskolin, significantly decreased the number of α-synuclein aggregates in the cortex of PINK1-KO rats, and is indistinguishable from WT rats. Overall, we show proof of concept that intranasal administration of CNS/CT-001 is a non-invasive, disease-modifying formulation for PD.

Nalfurafine Hydrochloride, a κ-Opioid Receptor Agonist, Induces Melanophagy via PKA Inhibition in B16F1 Cells.

Selective autophagy controls cellular homeostasis by degrading unnecessary or damaged cellular components. Melanosomes are specialized organelles that regulate the biogenesis, storage, and transport of melanin in melanocytes. However, the mechanisms underlying melanosomal autophagy, known as the melanophagy pathway, are poorly understood. To better understand the mechanism of melanophagy, we screened an endocrine-hormone chemical library and identified nalfurafine hydrochlorides, a κ-opioid receptor agonist, as a potent inducer of melanophagy. Treatment with nalfurafine hydrochloride increased autophagy and reduced melanin content in alpha-melanocyte-stimulating hormone (α-MSH)-treated cells. Furthermore, inhibition of autophagy blocked melanosomal degradation and reversed the nalfurafine hydrochloride-induced decrease in melanin content in α-MSH-treated cells. Consistently, treatment with other κ-opioid receptor agonists, such as MCOPPB or mianserin, inhibited excessive melanin production but induced autophagy in B16F1 cells. Furthermore, nalfurafine hydrochloride inhibited protein kinase A (PKA) activation, which was notably restored by forskolin, a PKA activator. Additionally, forskolin treatment further suppressed melanosomal degradation as well as the anti-pigmentation activity of nalfurafine hydrochloride in α-MSH-treated cells. Collectively, our data suggest that stimulation of κ-opioid receptors induces melanophagy by inhibiting PKA activation in α-MSH-treated B16F1 cells.

Opioid modulation of T-type Ca2+ channel-dependent neuritogenesis/neurite outgrowth through the prostaglandin E2/EP4 receptor/protein kinase A pathway in mouse dorsal root ganglion neurons

Irregular regeneration or inappropriate remodeling of the axons of the primary afferent neurons after peripheral nerve trauma could be associated with the development of neuropathic pain. We analyzed the molecular mechanisms for the neuritogenesis and neurite outgrowth caused by prostaglandin E2 (PGE2) in mouse dorsal root ganglion (DRG) neurons, and evaluated their opioid modulation. PGE2 in combination with IBMX, a phosphodiesterase inhibitor, caused neuritogenesis/neurite outgrowth in DRG cells, an effect abolished by a prostanoid EP4, but not EP2, receptor antagonist, and inhibitors of adenylyl cyclase or protein kinase A (PKA). Blockers of T-type Ca2+ channels (T-channels), that are responsible for window currents involving the sustained low-level Ca2+ entry at voltages near the resting membrane potentials and can be functionally upregulated by PKA, inhibited the neuritogenesis/neurite outgrowth caused by PGE2/IBMX or dibutylyl cyclic AMP, a PKA activator, in DRG neurons, an inhibitory effect mimicked by ZnCl2 and ascorbic acid that block Cav3.2, but not Cav3.1 or Cav3.3, T-channels. Morphine and DAMGO, μ-opioid receptor (MOR) agonists, suppressed the neuritogenesis and/or neurite outgrowth induced by PGE2/IBMX in DRG neurons and also DRG neuron-like ND7/23 cells, an effect reversed by naloxone or β-funaltrexamine, a selective MOR antagonist. Our data suggest that the EP4 receptor/PKA/Cav3.2 pathway is involved in the PGE2-induced neuritogenesis/neurite outgrowth in DRG neurons, which can be suppressed by MOR stimulation. We propose that MOR agonists including morphine in the early phase after peripheral nerve trauma might delay the axonal regeneration of the primary afferent neurons but prevent the development of neuropathic pain.

Intracellular sites of AQP2 S256 phosphorylation identified using inhibitors of the AQP2 recycling itinerary.

Vasopressin (VP)-regulated aquaporin-2 (AQP2) trafficking between cytoplasmic vesicles and the plasma membrane of kidney principal cells is essential for water homeostasis. VP affects AQP2 phosphorylation at several serine residues in the COOH-terminus; among them, serine 256 (S256) appears to be a major regulator of AQP2 trafficking. Mutation of this serine to aspartic acid, which mimics phosphorylation, induces constitutive membrane expression of AQP2. However, the intracellular location(s) at which S256 phosphorylation occurs remains elusive. Here, we used strategies to block AQP2 trafficking at different cellular locations in LLC-PK1 cells and monitored VP-stimulated phosphorylation of S256 at these sites by immunofluorescence and Western blot analysis with phospho-specific antibodies. Using methyl-β-cyclodextrin, cold block or bafilomycin, and taxol, we blocked AQP2 at the plasma membrane, in the perinuclear trans-Golgi network, and in scattered cytoplasmic vesicles, respectively. Regardless of its cellular location, VP induced a significant increase in S256 phosphorylation, and this effect was not dependent on a functional microtubule cytoskeleton. To further investigate whether protein kinase A (PKA) was responsible for S256 phosphorylation in these cellular compartments, we created PKA-null cells and blocked AQP2 trafficking using the same procedures. We found that S256 phosphorylation was no longer increased compared with baseline, regardless of AQP2 localization. Taken together, our data indicate that AQP2 S256 phosphorylation can occur at the plasma membrane, in the trans-Golgi network, or in cytoplasmic vesicles and that this event is dependent on the expression of PKA in these cells.NEW & NOTEWORTHY Phosphorylation of aquaporin-2 by PKA at serine 256 (S256) occurs in various subcellular locations during its recycling itinerary, suggesting that the protein complex necessary for AQP2 S256 phosphorylation is present in these different recycling stations. Furthermore, we showed, using PKA-null cells, that PKA activity is required for vasopressin-induced AQP2 phosphorylation. Our data reveal a complex spatial pattern of intracellular AQP2 phosphorylation at S256, shedding new light on the role of phosphorylation in AQP2 membrane accumulation.

Defective synaptic plasticity in a model of Coffin-Lowry syndrome is rescued by simultaneously targeting PKA and MAPK pathways.

Empirical and computational methods were combined to examine whether individual or dual-drug treatments can restore the deficit in long-term synaptic facilitation (LTF) of the Aplysia sensorimotor synapse observed in a cellular model of Coffin-Lowry syndrome (CLS). The model was produced by pharmacological inhibition of p90 ribosomal S6 kinase (RSK) activity. In this model, coapplication of an activator of the mitogen-activated protein kinase (MAPK) isoform ERK and an activator of protein kinase A (PKA) resulted in enhanced phosphorylation of RSK and enhanced LTF to a greater extent than either drug alone and also greater than their additive effects, which is termed synergism. The extent of synergism appeared to depend on another MAPK isoform, p38 MAPK. Inhibition of p38 MAPK facilitated serotonin (5-HT)-induced RSK phosphorylation, indicating that p38 MAPK inhibits activation of RSK. Inhibition of p38 MAPK combined with activation of PKA synergistically activated both ERK and RSK. Our results suggest that cellular models of disorders that affect synaptic plasticity and learning, such as CLS, may constitute a useful strategy to identify candidate drug combinations, and that combining computational models with empirical tests of model predictions can help explain synergism of drug combinations.

AKAP13 Enhances CREB1 Activation by FSH in Granulosa Cells.

Granulosa cells (GCs) must respond appropriately to follicle-stimulating hormone (FSH) for proper follicle maturation. FSH activates protein kinase A (PKA) leading to phosphorylation of the cyclic AMP response element binding protein-1 (CREB1). We identified a unique A-kinase anchoring protein (AKAP13) containing a Rho guanine nucleotide exchange factor (RhoGEF) region that was induced in GCs during folliculogenesis. AKAPs are known to coordinate signaling cascades, and we sought to evaluate the role of AKAP13 in GCs in response to FSH. Aromatase reporter activity was increased in COV434 human GCs overexpressing AKAP13. Addition of FSH, or the PKA activator forskolin, significantly enhanced this activity by 1.5- to 2.5-fold, respectively (p < 0.001). Treatment with the PKA inhibitor H89 significantly reduced AKAP13-dependent activation of an aromatase reporter (p = 0.0067). AKAP13 physically interacted with CREB1 in co-immunoprecipitation experiments and increased the phosphorylation of CREB1. CREB1 phosphorylation increased after FSH treatment in a time-specific manner, and this effect was reduced by siRNA directed against AKAP13 (p = 0.05). CREB1 activation increased by 18.5-fold with co-expression of AKAP13 in the presence of FSH (p < 0.001). Aromatase reporter activity was reduced by inhibitors of the RhoGEF region, C3 transferase and A13, and greatly enhanced by the RhoGEF activator, A02. In primary murine and COV43 GCs, siRNA knockdown of Akap13/AKAP13 decreased aromatase and luteinizing hormone receptor transcripts in cells treated with FSH, compared with controls. Collectively, these findings suggest that AKAP13 may function as a scaffolding protein in FSH signal transduction via an interaction with CREB, resulting in phosphorylation of CREB.

Locomotion activates PKA through dopamine and adenosine in striatal neurons.

The canonical model of striatal function predicts that animal locomotion is associated with the opposing regulation of protein kinase A (PKA) in direct and indirect pathway striatal spiny projection neurons (SPNs) by dopamine1-7. However, the precise dynamics of PKA in dorsolateral SPNs during locomotion remain to be determined. It is also unclear whether other neuromodulators are involved. Here we show that PKA activity in both types of SPNs is essential for normal locomotion. Using two-photon fluorescence lifetime imaging8-10 of a PKA sensor10 through gradient index lenses, we measured PKA activity within individual SPNs of the mousedorsolateral striatum during locomotion. Consistent with the canonical view, dopamine activated PKA activity in direct pathway SPNs during locomotion through the dopamine D1 receptor. However, indirect pathway SPNs exhibited a greater increase in PKA activity, which was largely abolished through the blockade of adenosine A2A receptors. In agreement with these results, fibre photometry measurements of an adenosine sensor11 revealed an acute increase in extracellular adenosine during locomotion. Functionally, antagonism of dopamine or adenosine receptors resulted in distinct changes in SPN PKA activity, neuronal activity and locomotion. Together, our results suggest that acute adenosine accumulation interplays with dopamine release to orchestrate PKA activity in SPNs and proper striatal function during animal locomotion.

Resistin impairs mitochondrial homeostasis via cyclase-associated protein 1-mediated fission, leading to obesity-induced metabolic diseases

ObjectiveOne of the suggested mechanisms of obesity-induced insulin resistance is mitochondrial dysfunction in target tissues such as skeletal muscle. In our study, we examined whether resistin, an adipokine associated with obesity-mediated insulin resistance, induced metabolic disorders by impairing mitochondrial homeostasis. MethodsThe morphology and function of mitochondria of skeletal muscle were examined in resistin-knockout and humanized resistin mice that were subjected to high-fat diet for 3 months. Morphology was examined by transmission electron microscopy. Mitochondria bioenergetics of skeletal muscle were evaluated using a Seahorse XF96 analyzer. Human skeletal myoblasts were used for in vitro studies on signaling mechanisms in responses to resistin. ResultsA high-fat diet in humanized resistin mice increased fragmented and shorter mitochondria in the skeletal muscle, whereas resistin-knockout mice had healthy normal mitochondria. In vitro studies showed that human resistin treatment impaired mitochondrial homeostasis by inducing mitochondrial fission, leading to a decrease in ATP production and mitochondrial dysfunction. Induction of mitochondrial fission by resistin was accompanied by increased formation of mitochondria-associated ER membranes (MAM). At the same time, resistin induced up-regulation of the protein kinase A (PKA) pathway. This activation of PKA induced phosphorylation of Drp1 at serine 616, leading to Drp1 activation and subsequent induction of mitochondrial fission. The key molecule that mediated human resistin-induced mitochondrial fission was adenylyl cyclase-associated protein 1 (CAP1), which was reported as a bona fide receptor for human resistin. Moreover, our newly developed biomimetic selective blocking peptide could repress human resistin-mediated mitochondrial dysfunction. High-fat diet-fed mice showed lower exercise capacity and higher insulin resistance, which was prevented by a novel peptide to block the binding of resistin to CAP1 or in the CAP1-knockdown mice. ConclusionsOur study demonstrated that human resistin induces mitochondrial dysfunction by inducing abnormal mitochondrial fission. This result suggests that the resistin-CAP1 complex could be a potential therapeutic target for the treatment of obesity-related metabolic diseases such as diabetes and cardiometabolic diseases.

Fear extinction induced by activation of PKA ameliorates anxiety-like behavior in PTSD mice

Prolonged exposure (PE) therapy aiming to promote fear extinction is a useful treatment for post-traumatic stress disorder (PTSD). However, the mechanisms underlying fear extinction and effective methods used to promote fear extinction in PTSD are still lacking. In this study, we displayed dysfunctions of cyclic adenosine 3,5-monophosphate (cAMP)-protein kinase A (PKA), protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and calcium signaling in peripheral serum of PTSD patients using bioinformatics analysis. Later, we confirmed the dysfunctions of cAMP-PKA, AKT/mTOR and calcium signaling in the hippocampus of PTSD mice. Moreover, the reduction of calpain1 in the hippocampus enhanced fear memory acquisition. Single activation of PKA by systemic application of rolipram (ROL) or meglumine cyclic adenylate (M-cAMP) before re-exposure promoted fear extinction and improved anxiety-like behavior in PTSD mice. Moreover, systemic application of ROL before re-exposure improved hippocampal brain-derived neurotrophic factor (BDNF)/tyrosine kinase receptor B (TrkB) signaling and calpain1/AKT/mTOR signaling. Interestingly, the effects of activation of PKA could be partially blocked by TrkB antagonist, ANA-12 and mTOR inhibitor, RAPA. Finally, intranasal administration of ROL could also adjust the abnormality of fear memory and improve anxiety-like behaviors in PTSD mice. Collectively, activation of PKA could promote fear extinction, which correlated with the reduction of anxiety-like behavior. The mechanisms were related to the BDNF/TrkB and calpain1/AKT/mTOR signaling pathways. PKA activation might be a useful complementary therapy for PE in the symptom elimination of PTSD.

A grooved porous hydroxyapatite scaffold induces osteogenic differentiation via regulation of PKA activity by upregulating miR-129-5p expression.

Hydroxyapatite scaffolds with different morphologies have been widely used in bone tissue engineering. Moreover, microRNAs (miRNAs) have been proven to be extensively involved in regulating bone regeneration. We developed grooved porous hydroxyapatite (HAG) scaffolds with good osteogenic efficiency. However, little is known about the role of miRNAs in HAG scaffold-mediated promotion of bone regeneration. The objective of this study was to reveal the mechanism from the perspective of differential miRNA expression. Scanning electron microscopy (SEM) was used to perform the coculture of cells and scaffolds. The miRNA profiles were generated by a microarray assay. A synthetic miR-129-5p mimic and inhibitor were used for overexpression or inhibition. The expression of osteogenic marker mRNAs and proteins was detected by quantitative real-time PCR (qRT-PCR), Western blotting, and immunofluorescence. An ALP activity kit and alizarin red staining (ARS) were used to measure ALP activity and mineral deposition formation. Cell migration ability was examined by wound healing and transwell assays. Protein kinase A (PKA) activity was measured by enzyme-linked immunosorbent assay (ELISA) after miR-129-5p transfection. Target genes were identified by a dual-luciferase reporter assay. H89 preculture evaluated the cross talk between miR-129-5p and PKA activity. Heterotopic implantation models, hematoxylin-eosin (HE), immunohistochemistry staining, and micro-CT were used to evaluate miR-129-5p osteogenesis in vivo. miRNAs were differentially expressed during osteogenic differentiation induced by HAG in vitro and in vivo. miR-129-5p was the only highly expressed miRNA both in vitro and in vivo. miR-129-5p overexpression promoted osteoblast differentiation and cell migration, while its inhibition weakened the effect of HAG. Moreover, miR-129-5p activated PKA to regulate the phosphorylation of β-catenin and cAMP-response element binding protein (CREB) by inhibiting cAMP-dependent protein kinase inhibitor alpha (Pkia). H89 prevented the effects of miR-129-5p on osteogenic differentiation and cell migration. HE, immunohistochemistry staining and micro-CT results showed that miR-129-5p promoted in vivo osteogenesis of the HAG scaffold. The HAG scaffold activates Pka by upregulating miR-129-5p and inhibiting Pkia, resulting in CREB-dependent transcriptional activation and accumulation of β-catenin and promoting osteogenic marker expression.

MHY4571, a novel diarylcyclohexanone derivative, exerts anti-cancer activity by regulating the PKA-cAMP-response element-binding protein pathway in squamous cell lung cancer

BackgroundThe protein kinase A (PKA)/cAMP response element-binding protein (CREB) has been suggested to be related to the inhibition of the proliferation of non-small cell lung cancer (NSCLC) cells. This study aimed to investigate the efficacy of a novel diarylcyclohexanone derivative, MHY4571, in regulating the PKA-CREB pathway and to study its anti-tumor role in squamous NSCLC.MethodsWe designed MHY4571 as a novel PKA inhibitor with acceptable in silico ADME properties and tested it in vitro in lung cancer cell lines and in vivo in xenograft and orthotopic mouse models of squamous cell lung carcinoma.ResultsMHY4571 inhibited PKA activity (> 70% inhibition) and suppressed the expression of p-PKA and p-CREB dose-dependently. MHY4571 treatment reduced lung cancer cell viability and promoted caspase 3-dependent apoptotic cell death. Orally administered MHY4571 significantly suppressed lung tumor growth in xenograft and orthotopic mouse models. PKA catalytic subunit alpha-silencing by siRNA (siPKA) strongly attenuated CREB phosphorylation; siCREB did not alter PKA protein levels or its phosphorylation, suggesting that PKA is an upstream regulator of CREB activity. MHY4571 acted synergistically with cisplatin (on co-treatment) to induce apoptotic cell death in lung cancer cells.ConclusionsOur results imply that MHY4571 may be a potential drug candidate for squamous cell lung cancer treatment.