Phosphatase 1B Research Articles

AMPA Receptor Antagonists Facilitate NEDD4-2-Mediated GRIA1 Ubiquitination by Regulating PP2B-ERK1/2-SGK1 Pathway in Chronic Epilepsy Rats.

The neural precursor cell expressed by developmentally downregulated gene 4-2 (NEDD4-2) is a ubiquitin E3 ligase that has a high affinity toward binding and ubiquitinating glutamate ionotropic receptor α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type subunit 1 (GRIA1, also referred to GluR1 or GluA1). Since dysregulation of GRIA1 surface expression is relevant to the responsiveness to AMPA receptor (AMPAR) antagonists (perampanel and GYKI 52466) in chronic epilepsy rats, it is likely that NEDD4-2 may be involved in the pathogenesis of intractable epilepsy. However, the role of NEDD4-2-mediated GRIA1 ubiquitination in refractory seizures to AMPAR antagonists is still unknown. In the present study, both AMPAR antagonists recovered the impaired GRIA1 ubiquitination by regulating protein phosphatase 2B (PP2B)-extracellular signal-regulated kinase 1/2 (ERK1/2)-serum and glucocorticoid-regulated kinase 1 (SGK1)-NEDD4-2 signaling pathway in responders (whose seizure activities are responsive to AMPAR), but not non-responders (whose seizure activities were uncontrolled by AMPAR antagonists). In addition, cyclosporin A (CsA, a PP2B inhibitor) co-treatment improved the effects of AMPAR antagonists in non-responders, independent of AKT signaling pathway. Therefore, our findings suggest that dysregulation of PP2B-ERK1/2-SGK1-NEDD4-2-mediated GRIA1 ubiquitination may be responsible for refractory seizures and that this pathway may be a potential therapeutic target for improving the treatment of intractable epilepsy in response to AMPAR antagonists.

Retraction Note: Herbal formula GAPT prevents beta amyloid deposition induced Ca2+/Calmodulin-dependent protein kinase II and Ca2+/Calmodulin-dependent protein phosphatase 2B imbalance in APPV717I mice

Background Synaptic dysfunction is one of the pathological characteristics of Alzheimer's disease (AD), which is directly related to the progressive decline of cognitive function. CaMKII and CaN have been found to play important roles in memory processes and synaptic transmission. So present study aimed to elucidate relationships between CaMKII, CaN and cognitive decline in APPV717I mice, and to reveal whether the cognitive improving effects of GAPT is conducted through rebalance CaMKII and CaN.

Protein Phosphatase 2B Dual Function Facilitates Synaptic Integrity and Motor Learning.

Protein phosphatase 2B (PP2B) is critical for synaptic plasticity and learning, but the molecular mechanisms involved remain unclear. Here we identified different types of proteins that interact with PP2B, including various structural proteins of the postsynaptic densities (PSDs) of Purkinje cells (PCs) in mice. Deleting PP2B reduced expression of PSD proteins and the relative thickness of PSD at the parallel fiber to PC synapses, whereas reexpression of inactive PP2B partly restored the impaired distribution of nanoclusters of PSD proteins, together indicating a structural role of PP2B. In contrast, lateral mobility of surface glutamate receptors solely depended on PP2B phosphatase activity. Finally, the level of motor learning covaried with both the enzymatic and nonenzymatic functions of PP2B. Thus, PP2B controls synaptic function and learning both through its action as a phosphatase and as a structural protein that facilitates synapse integrity.SIGNIFICANCE STATEMENT Phosphatases are generally considered to serve their critical role in learning and memory through their enzymatic operations. Here, we show that protein phosphatase 2B (PP2B) interacts with structural proteins at the synapses of cerebellar Purkinje cells. Differentially manipulating the enzymatic and structural domains of PP2B leads to different phenotypes in cerebellar learning. We propose that PP2B is crucial for cerebellar learning via two complementary actions, an enzymatic and a structural operation.

Disruption of FOXO3a-miRNA feedback inhibition of IGF2/IGF-1R/IRS1 signaling confers Herceptin resistance in HER2-positive breast cancer

Resistance to Herceptin represents a significant challenge for successful treatment of HER2-positive breast cancer. Here, we show that in Herceptin-sensitive cells, FOXO3a regulates specific miRNAs to control IGF2 and IRS1 expression, retaining basic IGF2/IGF-1R/IRS1 signaling. The basic activity maintains expression of PPP3CB, a subunit of the serine/threonine-protein phosphatase 2B, to restrict FOXO3a phosphorylation (p-FOXO3a), inducing IGF2- and IRS1-targeting miRNAs. However, in Herceptin-resistant cells, p-FOXO3a levels are elevated due to transcriptional suppression of PPP3CB, disrupting the negative feedback inhibition loop formed by FOXO3a and the miRNAs, thereby upregulating IGF2 and IRS1. Moreover, we detect significantly increased IGF2 in blood and IRS1 in the tumors of breast cancer patients with poor response to Herceptin-containing regimens. Collectively, we demonstrate that the IGF2/IGF-1R/IRS1 signaling is aberrantly activated in Herceptin-resistant breast cancer via disruption of the FOXO3a-miRNA negative feedback inhibition. Such insights provide avenues to identify predictive biomarkers and effective strategies overcoming Herceptin resistance.

Serine/Threonine Phosphatases in LTP: Two B or Not to Be the Protein Synthesis Blocker-Induced Impairment of Early Phase.

Dephosphorylation of target proteins at serine/threonine residues is one of the most crucial mechanisms regulating their activity and, consequently, the cellular functions. The role of phosphatases in synaptic plasticity, especially in long-term depression or depotentiation, has been reported. We studied serine/threonine phosphatase activity during the protein synthesis blocker (PSB)-induced impairment of long-term potentiation (LTP). Established protein phosphatase 2B (PP2B, calcineurin) inhibitor cyclosporin A prevented the LTP early phase (E-LTP) decline produced by pretreatment of hippocampal slices with cycloheximide or anisomycin. For the first time, we directly measured serine/threonine phosphatase activity during E-LTP, and its significant increase in PSB-treated slices was demonstrated. Nitric oxide (NO) donor SNAP also heightened phosphatase activity in the same manner as PSB, and simultaneous application of anisomycin + SNAP had no synergistic effect. Direct measurement of the NO production in hippocampal slices by the NO-specific fluorescent probe DAF-FM revealed that PSBs strongly stimulate the NO concentration in all studied brain areas: CA1, CA3, and dentate gyrus (DG). Cyclosporin A fully abolished the PSB-induced NO production in the hippocampus, suggesting a close relationship between nNOS and PP2B activity. Surprisingly, cyclosporin A alone impaired short-term plasticity in CA1 by decreasing paired-pulse facilitation, which suggests bi-directionality of the influences of PP2B in the hippocampus. In conclusion, we proposed a minimal model of signaling events that occur during LTP induction in normal conditions and the PSB-treated slices.

Hypoxia‐Induced TIMAP Upregulation Promotes Tumor Angiogenesis by Attenuating ALK1‐Mediated TIMAP Repression

Introduction TIMAP (TGF-β1 inhibited membrane associated protein) is an endothelial cell (EC)-predominant inhibitory protein phosphatase 1β (PP1β) regulatory subunit in the myosin phosphatase targeting (MYPT) family (J. Biol. Chem. 2294:13280, 2019). TIMAP promotes proliferation and survival of EC and enhances in vitro sprouting angiogenesis (Am. J. Physiol. - Renal Physiol. 307: F623, 2014). Hypothesis Hypoxia stimulates TIMAP expression in EC, enhancing in vivo tumor angiogenesis. Methods Mammary adenocarcinoma cells (E0771, syngeneic for C57BL/6 mice) were injected into the mammary glands of five pairs of female TIMAP+/+ / TIMAP-/- C57BL/6 mice at 6-8 weeks of age. For each pair, mice were euthanized on the day the tumor diameter exceeded 1.5 cm in one mouse. Tumor weight, mean tumor diameter, and tumor vascular density were then quantified. For quantification of vascular density, the EC markers were PECAM1 and Tie-2 for fluorescence microscopy (IF) and western blot (WB) analysis, respectively. TIMAP+/+ mice were also exposed to progressive, chronic hypoxia by reducing the ambient O2 concentration from 18 to 10 % over two weeks and then maintaining it at 10% for 3 weeks. Lung tissue was then evaluated for TIMAP abundance by WB analysis, and TIMAP localization was determined by IF. Finally, the mechanism regulating TIMAP expression was evaluated in cultured EC in the presence of 21% and 1% O2 with or without TGF-β pathway activation or inhibition. Results Tumor size was similar in TIMAP+/+ and TIMAP-/- mice through days 8-10 after injection of E0771 cells. In 4 of 5 TIMAP-/-, but not TIMAP+/+ mice, the tumors then eroded partially through the skin and regressed gradually. At the time of euthanasia tumor size (Fig. 1A), weight (Fig. 1B) and blood vessel density (Fig.1C, D&E) were significantly lower in TIMAP-/- compared to TIMAP+/+ mice. In the chronic hypoxia model, TIMAP protein abundance in lung lysates was significantly higher in hypoxic compared to control mice, and IF using anti-TIMAP and anti-PECAM1 antibodies revealed that the TIMAP protein was restricted to lung EC. In cultured EC, HIF-1α and TIMAP protein abundance were significantly higher in EC exposed to 1% than 21% O2, (Fig. 1F,G). The effect of 1% O2 on TIMAP was only observed in the presence of fresh 1% serum or BMP-9 (1-10 ng/ml), while the hypoxia-induced increase in HIF-1α abundance was independent of serum or BMP-9. In the absence of hypoxia 1% serum and BMP-9 strongly stimulated Smad 1/5/8 but not Smad 2/3 phosphorylation and significantly reduced TIMAP protein levels. The ALK1 inhibitor K02288 but not the ALK 5 inhibitor LY-364947 blocked 1% serum and BMP-9 stimulated Smad 1/5/8 phosphorylation and raised the EC TIMAP abundance. Notably, hypoxia mimicked the ALK1 inhibitor, significantly blunting 1% serum and BMP-9-stimulated Smad 1/5/8 phosphorylation. Conclusion Hence, hypoxia induces expression of the pro-angiogenic PP1β inhibitor TIMAP due, at least in part, to inhibition of BMP-9/ALK1-mediated repression of TIMAP. Furthermore, TIMAP is necessary for effective tumor angiogenesis in vivo. We therefore speculate that tumor hypoxia stimulates EC TIMAP expression by inhibiting the ALK1 pathway.

Transient receptor potential vanilloid 2 mediates the inhibitory effect of far-infrared irradiation on adipogenic differentiation of tonsil-derived mesenchymal stem cells

AimsFar-infrared (FIR) irradiation inhibits adipogenic differentiation of tonsil-derived mesenchymal stem cells (TMSCs) by activating Ca2+-dependent protein phosphatase 2B (PP2B), but it stimulates osteogenic differentiation in a PP2B-independent pathway. We investigated the potential involvement of transient receptor potential vanilloid (TRPV) channels, a well-known Ca2+-permeable channel, in the effects of FIR irradiation on adipogenic or osteogenic differentiation of TMSCs. MethodsTMSCs, in the absence or presence of activators or inhibitors, were exposed to FIR irradiation followed by adipogenic or osteogenic differentiation, which was assessed using Oil red O or Alizarin red S staining, respectively. RT-PCR, qRT-PCR, and Western blotting were used to determine gene and protein expression of calcium channels and adipocyte-specific markers. ResultsTreatment with the calcium ionophore ionomycin simulated the inhibitory effect of FIR irradiation on adipogenic differentiation but had no effect on osteogenic differentiation, indicating the involvement of intracellular Ca2+ in adipogenic differentiation. Inhibition of pan-TRP channels using ruthenium red reversed the FIR irradiation-induced inhibition of adipogenic differentiation. Among the TRP channels tested, inhibition of the TRPV2 channel by tranilast or siRNA against TRPV2 attenuated the inhibitory effect of FIR irradiation on adipogenic differentiation, accompanied by a decrease in intracellular Ca2+ levels. By contrast, activation of the TRPV2 channel by probenecid simulated FIR irradiation-induced inhibition of adipogenic differentiation. Expectedly, the stimulatory effect of FIR irradiation on osteogenic differentiation was independent of the TRPV2 channel. ConclusionOur data demonstrate that the TRPV2 channel is a sensor/receptor for the inhibited adipogenic differentiation of TMSCs associated with FIR irradiation.

AKAP79/150 coordinates leptin-induced PKA signaling to regulate KATP channel trafficking in pancreatic β-cells

The adipocyte hormone leptin regulates glucose homeostasis both centrally and peripherally. A key peripheral target is the pancreatic β-cell, which secretes insulin upon glucose stimulation. Leptin is known to suppress glucose-stimulated insulin secretion by promoting trafficking of KATP channels to the β-cell surface, which increases K+ conductance and causes β-cell hyperpolarization. We have previously shown that leptin-induced KATP channel trafficking requires protein kinase A (PKA)-dependent actin remodeling. However, whether PKA is a downstream effector of leptin signaling or PKA plays a permissive role is unknown. Using FRET-based reporters of PKA activity, we show that leptin increases PKA activity at the cell membrane and that this effect is dependent on N-methyl-D-aspartate receptors, CaMKKβ, and AMPK, which are known to be involved in the leptin signaling pathway. Genetic knockdown and rescue experiments reveal that the increased PKA activity upon leptin stimulation requires the membrane-targeted PKA-anchoring protein AKAP79/150, indicating that PKA activated by leptin is anchored to AKAP79/150. Interestingly, disrupting protein phosphatase 2B (PP2B) anchoring to AKAP79/150, known to elevate basal PKA signaling, leads to increased surface KATP channels even in the absence of leptin stimulation. Our findings uncover a novel role of AKAP79/150 in coordinating leptin and PKA signaling to regulate KATP channel trafficking in β-cells, hence insulin secretion. The study further advances our knowledge of the downstream signaling events that may be targeted to restore insulin secretion regulation in β-cells defective in leptin signaling, such as those from obese individuals with type 2 diabetes.

The CnB1 p.D102A variant is linked to dilated cardiomyopathy via impaired Calcineurin activity

Background:The role of calcineurin (protein phosphatase 2B (PP2B)) in the pathogenesis of human dilated cardiomyopathy (DCM) has not been fully elucidated. We determined the potential involvement of calcineurin in the pathogenesis of DCM caused by mutations in CnB1, a subunit of calcineurin.Methods: By whole-exome sequencing, we identified a new CnB1 variant in a Han Chinese proband with cardiomyopathy from a 3-generation family with 2 normal individuals and 3 individuals with familial dilated cardiomyopathy. The potential pathogenic variant was validated by Sanger sequencing. We performed functional and mechanistic experiments in a CnB1-knockin (KI) mouse model and at the cellular level.Results: We detected a rare heterozygous CnB1 variant (p.D102A)in a proband with dilated cardiomyopathy. This variant was localized to the EF hand 3 region of CnB1, where no variants have been previously reported. KI mice harboring the p.D102A variant exhibited decreased cardiac function and cardiac dilatation. Immunoblotting, RT-PCR and immunofluorescence results showed decreased cardiomyocyte size and heart failure-related protein expression. A calcineurin activity assay demonstrated decreased calcineurin activity in the KI mice, accompanied by the decreased ability of CnB1 to bind CnA.Conclusions: CnB1 p.D102A is a disease-associated variantthat confers susceptibility to cardiac dilatation. This variant is associated with impaired calcineurin activity and a subsequent decrease in the ability of CnB1 to bind CnA.

Di-(2-ethylhexyl) phthalate limits the pleiotropic effects of statins in chronic kidney disease patients undergoing dialysis and endothelial cells

The level of di-(2-ethylhexyl) phthalate (DEHP) is elevated in chronic kidney disease patients undergoing dialysis. However, statins are unable to reduce the cardiovascular events in chronic dialysis patients. In this study, we investigated the effects of DEHP on statin-conferred pleiotropic effects and the underlying molecular mechanism in peritoneal dialysis (PD) patients and endothelial cells (ECs). In PD patients with serum DEHP level ≥0.0687 μg/mL, statin treatment was not associated with lower risk of cardiovascular disease. In ECs, exposure to DEHP abrogated the simvastatin-induced NO bioavailability and EC-related functions. Additionally, DEHP abolished the anti-inflammatory effect of simvastatin on the tumor necrosis factor α-induced upregulation of adhesion molecules and monocyte adhesion to ECs. Mechanistically, DEHP blunted the activation of transient receptor potential vanilloid type 1 (TRPV1), which is required for NO production by simvastatin in ECs. Notably, DEHP increased the activity and expression of protein phosphatase 2B (PP2B), a negative regulator of TRPV1 activity. The effect of DEHP on PP2B activation was mediated by the activation of the NADPH oxidase/reactive oxygen species (NOX−ROS) pathway. Inhibition of PP2B activity by pharmacological antagonists prevented the inhibitory effects of DEHP on simvastatin-induced Ca2+ influx, NO bioavailability, and EC migration, proliferation, tube formation, and anti-inflammatory action. Collectively, DEHP activates the NOX−ROS−PP2B pathway, which in turns inhibits TRPV1/Ca2+-dependent signaling and abrogates the statin-conferred pleiotropic protection in ECs.

Binding of Filamentous Actin to CaMKII as Potential Regulation Mechanism of Bidirectional Synaptic Plasticity by \u03b2 CaMKII in Cerebellar Purkinje Cells

Calcium-calmodulin dependent protein kinase II (CaMKII) regulates many forms of synaptic plasticity, but little is known about its functional role during plasticity induction in the cerebellum. Experiments have indicated that the β isoform of CaMKII controls the bidirectional inversion of plasticity at parallel fibre (PF)-Purkinje cell (PC) synapses in cerebellar cortex. Because the cellular events that underlie these experimental findings are still poorly understood, we developed a simple computational model to investigate how β CaMKII regulates the direction of plasticity in cerebellar PCs. We present the first model of AMPA receptor phosphorylation that simulates the induction of long-term depression (LTD) and potentiation (LTP) at the PF-PC synapse. Our simulation results suggest that the balance of CaMKII-mediated phosphorylation and protein phosphatase 2B (PP2B)-mediated dephosphorylation of AMPA receptors can determine whether LTD or LTP occurs in cerebellar PCs. The model replicates experimental observations that indicate that β CaMKII controls the direction of plasticity at PF-PC synapses, and demonstrates that the binding of filamentous actin to CaMKII can enable the β isoform of the kinase to regulate bidirectional plasticity at these synapses.

Transient receptor potential vanilloid 2 mediates the inhibitory effect of far-infrared irradiation on adipogenic differentiation of tonsil-derived mesenchymal stem cells

Background & Aim Far-infrared (FIR) irradiation inhibits adipogenic differentiation of tonsil-derived mesenchymal stem cells (TMSCs) by activating protein phosphatase 2B through enhancing intracellular Ca2+ mobilization, but its upsteam mechanism remains unclear. Transient receptor potential vanilloid (TRPV) is a well-known calcium-permeable channel located in the cell membrane. It has been reported that certain isotypes of TRPV could regulate adipogenesis in (pre)adipocytes. Here, we investigated whether or how the TRPV channel is involved in the inhibitory effect of FIR irradiation on the adipogenic differentiation of TMSCs. Methods, Results & Conclusion Methods TMSCs were exposed to FIR irradiation with or without TRPV inhibitors at room temperature for 30 min followed by adipogenic differentiation for 14 days. At the end of the differentiation, the degree of adipogenesis was assessed by staining lipid droplets with Oil red O solution, and by measuring the protein expressions of adipocyte-specific markers (peroxisome proliferator-activated receptor γ and fatty acid-binding protein 4) using Western blotting. Total RNA was extracted from TMSCs and mRNA transcript levels were measured by reverse transcript (RT)-PCR analysis. Results Exposure to FIR irradiation or a calcium ionophore ionomycin inhibited adipogenic differentiation of TMSCs. Treatment with ruthenium red, a pan-TRP channel inhibitor, reversed the FIR irradiation-inhibited adipogenesis. RT-PCR analysis revealed that TMSCs expressed all the TRPV channels except TRPV5. Based on the previous studies showing an involvement of TRPV1, 2, and 4 in adipogenesis, we tested specific inhibitors of these three TRPV isotypes and found that among those tested, only tranilast, a specific TRPV2 inhibitor, significantly attenuated the effects of FIR irradiation, such as increase of intracellular Ca2+ levels and inhibition of adipogenic differentiation of the TMSCs. When the TMSCs were transfected with small interfering RNA against TRPV2 to silence the TRPV2 gene, the observed effects of FIR irradiation on adipogenic differentiation were also completely reversed. Additionally, we also found that the probenecid, a TRPV2 activator, decreased the adipogenic differentiation of TMSCs. Conclusion Our data demonstrate that FIR irradiation increases intracellular Ca2+ levels through TRPV2 activation, thereby suppressing the adipogenic differentiation of TMSCs.

Lectin rich horsegram protein and myricetin activates insulin signaling – A study targeting PTP1β

Abstract Horsegram, a rich source of myricetin (Myr), exhibits an anti-hyperglycemic effect. We examined the individual and combinational effect of Myr and horsegram protein (HP) on protein tyrosine phosphatase 1β (PTP1β), and its effect on insulin signaling in high-fat-diet (HFD) and a single dose of streptozotocin (STZ) induced diabetic rat model. Myr, HP, and their combination, Myr + HP, were orally administered to diabetic animals for four weeks. Myr, HP, and Myr + HP were effective in reducing elevated blood glucose, HbA1c, fasting insulin, insulin resistance, oxidative stress markers, and proinflammatory cytokines. Myr ameliorated dyslipidemia and significantly reduced PTP1β activity; HP and Myr + HP were not as effective, although they suppressed the expression of PTP1β. Myr and HP treated groups had elevated levels of the phospho-insulin receptor (pIR), phospho-insulin receptor substrate1 (pIRS1), and phospho-Akt (pAkt), whereas, Myr + HP was not effective. Though myricetin and horsegram proteins ameliorate diabetic conditions, dietary intake of their combination reduced individual efficiency.

Brain sterol flux mediated by cytochrome P450 46A1 affects membrane properties and membrane-dependent processes.

Cytochrome P450 46A1 encoded by CYP46A1 catalyzes cholesterol 24-hydroxylation and is a CNS-specific enzyme that controls cholesterol removal and turnover in the brain. Accumulating data suggest that increases in cytochrome P450 46A1 activity in mouse models of common neurodegenerative diseases affect various, apparently unlinked biological processes and pathways. Yet, the underlying reason for these multiple enzyme activity effects is currently unknown. Herein, we tested the hypothesis that cytochrome P450 46A1-mediated sterol flux alters physico-chemical properties of the plasma membranes and thereby membrane-dependent events. We used 9-month-old 5XFAD mice (an Alzheimer’s disease model) treated for 6 months with the anti-HIV drug efavirenz. These animals have previously been shown to have improved behavioural performance, increased cytochrome P450 46A1 activity in the brain, and increased sterol flux through the plasma membranes. We further examined 9-month-old Cyp46a1−/− mice, which have previously been observed to have cognitive deficits and decreased sterol flux through brain membranes. Synaptosomal fractions from the brain of efavirenz-treated 5XFAD mice had essentially unchanged cholesterol levels as compared to control 5XFAD mice. However with efavirenz treatment in these mice, there were changes in the membrane properties (increased cholesterol accessibility, ordering, osmotic resistance and thickness) as well as total glutamate content and ability to release glutamate in response to mild stimulation. Similarly, the cholesterol content in synaptosomal fractions from the brain of Cyp46a1−/− mice was essentially the same as in wild-type mice but knockout of Cyp46a1 was associated with changes in membrane properties and glutamate content and its exocytotic release. Changes in Cyp46a1−/− mice were in the opposite direction to those observed in efavirenz-treated versus control 5XFAD mice. Incubation of synaptosomal fractions with the inhibitors of glycogen synthase kinase 3, cyclin-dependent kinase 5, protein phosphatase 1/2 A, and protein phosphatase 2B revealed that increased sterol flux in efavirenz-treated versus control 5XFAD mice affected the ability of all four enzymes to modulate glutamate release. In contrast, in Cyp46a1−/− versus wild-type mice, decreased sterol flux altered the ability of only cyclin-dependent kinase 5 and protein phosphatase 2B to regulate the glutamate release. Collectively, our results support cytochrome P450 46A1-mediated sterol flux as an important contributor to the fundamental properties of the membranes, protein phosphorylation and synaptic transmission. Also, our data provide an explanation of how one enzyme, cytochrome P450 46A1, can affect multiple pathways and processes and serve as a common potential target for several neurodegenerative disorders.

Small nucleolar RNA Sf-15 regulates proliferation and apoptosis of Spodoptera frugiperda Sf9 cells

BackgroundSmall nucleolar RNAs (snoRNAs) function in guiding 2′-O-methylation and pseudouridylation of ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs). In recent years, more and more snoRNAs have been found to play novel roles in mRNA regulation, such as pre-mRNA splicing or RNA editing. In our previous study, we found a silkworm C/D box snoRNA Bm-15 can interact with Notch receptor gene in vitro. To further study the function of Bm-15, we cloned its homolog Sf-15 from Spodoptera frugiperda and investigate the function of Sf-15 in Sf9 cells.ResultsWe showed that knocking down of Sf-15 can inhibit the proliferation, then induce apoptosis of insect S. frugiperda Sf9 cells, but the results were reversed when Sf-15 was overexpressed. De novo sequencing of transcriptome of Sf9 cells showed that the expression of 21 apoptosis-related genes were increased upon Sf-15 repression. Further analysis showed that a Ca2+-induced cell death pathway gene Cn (PPP3C, the serine/threonine-protein phosphatase 2B catalytic subunit), was significantly increased upon Sf-15 depression but decreased when Sf-15 was overexpressed, which indicated that Cn might be a potential target of Sf-15.ConclusionsWe conclude that C/D box snoRNA Sf-15 can participate in apoptosis through regulating the expression of Ca2+-induced cell death pathway gene Cn in Sf9 cells. This is the first time that we found snoRNAs exhibiting dual functions in insect, which reveals a novel layer of ncRNA modulation in cell growth and death.

Empagliflozin Contributes to Polyuria via Regulation of Sodium Transporters and Water Channels in Diabetic Rat Kidneys.

Besides lowering glucose, empagliflozin, a selective sodium-glucose cotransporter-2 (SGLT2) inhibitor, have been known to provide cardiovascular and renal protection due to effects on diuresis and natriuresis. However, the natriuretic effect of SGLT2 inhibitors has been reported to be transient, and long-term data related to diuretic change are sparse. This study was performed to assess the renal effects of a 12-week treatment with empagliflozin (3 mg/kg) in diabetic OLETF rats by comparing it with other antihyperglycemic agents including lixisenatide (10 μg/kg), a glucagon-like peptide receptor-1 agonist, and voglibose (0.6 mg/kg), an α-glucosidase inhibitor. At 12 weeks of treatment, empagliflozin-treated diabetic rats produced still high urine volume and glycosuria, and showed significantly higher electrolyte-free water clearance than lixisenatide or voglibose-treated diabetic rats without significant change of serum sodium level and fractional excretion of sodium. In empagliflozin-treated rats, renal expression of Na+-Cl- cotransporter was unaltered, and expressions of Na+/H+ exchanger isoform 3, Na+-K+-2Cl- cotransporter, and epithelial Na+ channel were decreased compared with control diabetic rats. Empagliflozin increased an expression of aquaporin (AQP)7 but did not affect AQP3 and AQP1 protein expressions in diabetic kidneys. Despite the increased expression in vasopressin V2 receptor, protein and mRNA levels of AQP2 in empagliflozin-treated diabetic kidneys were significantly decreased compared to control diabetic kidneys. In addition, empagliflozin resulted in the increased phosphorylation of AQP2 at S261 through the increased cyclin-dependent kinases 1 and 5 and protein phosphatase 2B. These results suggest that empagliflozin may contribute in part to polyuria via its regulation of sodium channels and AQP2 in diabetic kidneys.

Low-dose oral copper treatment changes the hippocampal phosphoproteomic profile and perturbs mitochondrial function in a mouse model of Alzheimer's disease

Excessive copper can cause neurotoxicity and contribute to the development of some neurological diseases; however, copper neurotoxicity and the potential mechanisms remain poorly understood. We used proteomics and phosphoproteomics to quantify protein changes in the hippocampus of wild-type and 3xTg-AD mice, both of which were treated at 6 months of age with 2 months of drinking water with or without added copper chloride (0.13 ppm concentration). A total of 3960 unique phosphopeptides (5290 phosphorylation sites) from 1406 phosphoproteins was identified. Differentially expressed phosphoproteins involved neuronal and synaptic function, transcriptional regulation, energy metabolism and mitochondrial function. In addition, low-dose copper treatment of wild-type mice decreased hippocampal mitochondrial copy number, mitochondrial biogenesis and disrupted mitochondrial dynamics; these changes were associated with increased hydrogen peroxide production (H2O2), reduced cytochrome oxidase activity and decreased ATP content. In 3xTg-AD mice, identical low-dose oral copper treatment increased axonal degeneration, which was associated with altered phosphorylation of Camk2α at T286 and phosphorylation of mitogen-activated protein kinase (ERK1/2), which involved long-term potentiation (LTP) signaling. Mitochondrial dysfunction was mainly related to changes in phosphorylation levels of glycogen synthase kinase-3 beta (GSK3β) and serine/threonine-protein phosphatase 2B catalytic subunit alpha isoform (Ppp3ca), which involved mitochondrial biogenesis signaling. In sum, low-dose oral copper treatment changes the phosphorylation of key hippocampal proteins involved in mitochondrial, synaptic and axonal integrity. These data showing that excess of copper speeds some early events of AD changes observed suggest that excess circulating copper has the potential to perturb brain function of wild-type mice and exacerbate neurodegenerative changes in a mouse model of AD.

Far-Infrared Irradiation Inhibits Adipogenic Differentiation and Stimulates Osteogenic Differentiation of Human Tonsil-Derived Mesenchymal Stem Cells: Role of Protein Phosphatase 2B.

Far-infrared (FIR) irradiation has been reported to exhibit various biological effects including improvement of cardiovascular function. However, its effect on the differentiation of stem cells has not been studied. Using tonsil-derived mesenchymal stem cells (TMSC), we examined whether and how FIR irradiation affects adipogenic or osteogenic differentiation. TMSC were exposed to FIR irradiation (3-25 μm wavelength) for various times (0, 30, or 60 min), and then adipogenic or osteogenic differentiation was induced for 14 days with its respective commercially available differentiation medium. At the end of the differentiation, the cells were stained using Oil red O or Alizarin red S solution, and the expression of differentiation-specific proteins was analyzed by western blotting. FIR irradiation did not alter cell viability or the expression of MSC-specific surface antigens (CD14, CD34, CD45, CD73, CD90, and CD105) in TMSC. However, FIR irradiation significantly inhibited adipogenic differentiation of TMSC, as evidenced by decreased Oil red O staining as well as protein expression of peroxisome proliferator-activated receptor γ and fatty acid binding protein 4. In contrast, FIR irradiation induced osteogenic differentiation, as evidenced by increased Alizarin red S staining as well as protein expression of osteocalcin and alkaline phosphatase. Treatment with heat alone did not inhibit the adipogenic differentiation of TMSC, suggesting that the inhibitory effect on adipogenic differentiation was not due to heat induced by FIR irradiation. However, heat alone did stimulate osteogenic differentiation, but to a lesser extent than FIR irradiation. Furthermore, FIR irradiation increased intracellular Ca²⁺ levels and the activity of protein phosphatase 2B (PP2B) in TMSC. Treatment with cyclosporin A, a specific PP2B inhibitor, reversed the inhibitory effect of FIR irradiation on adipogenic differentiation of TMSC, but had no effect on osteogenic differentiation. Our data demonstrate that FIR irradiation inhibits adipogenic differentiation but enhances osteogenic differentiation of TMSC; the inhibitory effect on adipogenic differentiation is non-thermal and mediated at least in part by activation of Ca²⁺-dependent PP2B.

Probing Mitochondrial Permeability Transition Pore Activity in Nucleated Cells and Platelets by High-Throughput Screening Assays Suggests Involvement of Protein Phosphatase 2B in Mitochondrial Dynamics.

Mitochondrial permeability transition pore (mPTP) formation is well documented in isolated mitochondria. However, convincing detection of mPTP in whole cells remains elusive. In this study, we describe a high-throughput assay for Ca2+-activated mPTP opening in platelets using HyperCyt flow cytometry. In addition, we demonstrate that in several nucleated cells, using multiple approaches, the detection of cyclophilin D-dependent mPTP opening is highly challenging. Results with the mitochondrial-targeted Ca2+-sensing green fluorescent protein (mito-Case12) suggest the involvement of protein phosphatase 2B (PP2B; calcineurin) in regulating mitochondrial dynamics. Our results highlight the danger of relying on cyclosporine A alone as a pharmacological tool, and the need for comprehensive studies of mPTP in the cell.

Modeling, Synthesis and Characterization of Phospho–Penta–Peptide derived from PKA RII Sub Unit: A Candidate Substrate for CaN Assay

Calcineurin (CaN) plays an important role in many pathological conditions like cancer, metabolic aberrations and neurodegenerative disorders. Till date contemporary methods to assay CaN uses p-nitrophenylphosphate (p-NPP) and specific protein phosphatases 2B inhibitor or use of phospho-peptide substrate derived from protein kinase A (PKA) regulatory sub unit type PKARII. Here we have modeled a series of small phospho peptides derived from PKARII and were docked by Molegro Virtual Docking software to find out the binding free enegy and to determine the affinity with modeled Calcineurin catalytic subunit A (CaN A). Further the candidate phospho–penta–peptide (PPP) derived from PKA regulatory sub unit type II was synthesized in house by employing Fmoc chemistry by solution phase method, purified over HPLC and characterized by mass spectrometry. This PPP was employed to assay CaN activity by Malachite Green method found to be a candidate substrate for CaN.