Increased Protein Phosphatase 2A Activity Research Articles

A prolyl oligopeptidase inhibitor reduces tau pathology in cellular models and in mice with tauopathy.

Tauopathies are neurodegenerative diseases that are characterized by accumulation of hyperphosphorylated tau protein, higher-order aggregates, and tau filaments. Protein phosphatase 2A (PP2A) is a major tau dephosphorylating phosphatase, and a decrease in its activity has been demonstrated in tauopathies, including Alzheimer's disease. Prolyl oligopeptidase is a serine protease that is associated with neurodegeneration, and its inhibition normalizes PP2A activity without toxicity under pathological conditions. Here, we assessed whether prolyl oligopeptidase inhibition could protect against tau-mediated toxicity in cellular models in vitro and in the PS19 transgenic mouse model of tauopathy carrying the human tau-P301S mutation. We show that inhibition of prolyl oligopeptidase with the inhibitor KYP-2047 reduced tau aggregation in tau-transfected HEK-293 cells and N2A cells as well as in human iPSC-derived neurons carrying either the P301L or tau-A152T mutation. Treatment with KYP-2047 resulted in increased PP2A activity and activation of autophagic flux in HEK-293 cells and N2A cells and in patient-derived iNeurons, as indicated by changes in autophagosome and autophagy receptor markers; this contributed to clearance of insoluble tau. Furthermore, treatment of PS19 transgenic mice for 1 month with KYP-2047 reduced tau burden in the brain and cerebrospinal fluid and slowed cognitive decline according to several behavioral tests. In addition, a reduction in an oxidative stress marker was seen in mouse brains after KYP-2047 treatment. This study suggests that inhibition of prolyl oligopeptidase could help to ameliorate tau-dependent neurodegeneration.

HIF-1α Causes LCMT1/PP2A Deficiency and Mediates Tau Hyperphosphorylation and Cognitive Dysfunction during Chronic Hypoxia.

Chronic hypoxia is a risk factor for Alzheimer's disease (AD), and the neurofibrillary tangle (NFT) formed by hyperphosphorylated tau is one of the two major pathological changes in AD. However, the effect of chronic hypoxia on tau phosphorylation and its mechanism remains unclear. In this study, we investigated the role of HIF-1α (the functional subunit of hypoxia-inducible factor 1) in tau pathology. It was found that in Sprague-Dawley (SD) rats, global hypoxia (10% O2, 6 h per day) for one month induced cognitive impairments. Meanwhile it induced HIF-1α increase, tau hyperphosphorylation, and protein phosphatase 2A (PP2A) deficiency with leucine carboxyl methyltransferase 1(LCMT1, increasing PP2A activity) decrease in the rats' hippocampus. The results were replicated by hypoxic treatment in primary hippocampal neurons and C6/tau cells (rat C6 glioma cells stably expressing human full-length tau441). Conversely, HIF-1α silencing impeded the changes induced by hypoxia, both in primary neurons and SD rats. The result of dual luciferase assay proved that HIF-1α acted as a transcription factor of LCMT1. Unexpectedly, HIF-1α decreased the protein level of LCMT1. Further study uncovered that both overexpression of HIF-1α and hypoxia treatment resulted in a sizable degradation of LCMT1 via the autophagy--lysosomal pathway. Together, our data strongly indicated that chronic hypoxia upregulates HIF-1α, which obviously accelerated LCMT1 degradation, thus counteracting its transcriptional expression. The increase in HIF-1α decreases PP2A activity, finally resulting in tau hyperphosphorylation and cognitive dysfunction. Lowering HIF-1α in chronic hypoxia conditions may be useful in AD prevention.

Abstract P1067: IRS Mediated Recruitment Of PI3Kgamma By Insulin Inhibits Beta-adrenergic Receptor Function

Insulin (INS) treatment results in impaired response to β-agonist isoproterenol (ISO) associated with phosphorylated β2-adrenergic receptor (β2AR) and impaired cAMP response. Although INS does not mediate dissociation of G-protein Gβγ subunits, yet surprisingly recruits phosphoinositide 3-kinase γ (PI3Kγ) to the plasma membrane. Correspondingly, knock-down of PI3Kγ (PI3Kγ KD) in HEK 293 or ablation in primary adult cardiomyocytes and fibroblasts (isolated from PI3Kγ knockout mice) abrogated β2AR phosphorylation reflecting a key role for PI3Kγ in retaining β2AR phosphorylation. Also, adult cardiomyocytes isolated from C57Bl6 mice showed significant loss of in vitro ISO-stimulated cardiomyocyte contraction upon INS pre-treatment which was remarkably preserved in the PI3Kγ knockout cardiomyocytes despite INS. As PI3Kγ is traditionally recruited to the βAR complex by the Gβγ subunits, we hypothesized that INS-mediates Gβγ-independent recruitment of PI3Kγ to the βAR complex underlying its dysfunction. INS stimulation is known to recruit insulin receptor substrate (IRS 1 & 2) to the receptor complex, and therefore, we tested whether PI3Kγ interacts with IRS 1/2 leading to Gβγ-independent recruitment of PI3Kγ to the β2AR complex. Immunoprecipitation PI3Kγ following INS showed that PI3Kγ interacts with IRS2 recruiting PI3Kγ to the β2AR complex bypassing the traditional Gβγ-dependent pathway. Since PI3Kγ KD resulted in loss of INS-mediated β2AR phosphorylation, we tested whether PI3Kγ inhibits protein phosphatase 2A (PP2A) function impairing β2AR de-phosphorylation. INS treatment resulted in significant loss of β2AR-associated PP2A activity which was rescued in PI3Kγ KD cells showing that PI3Kγ impairs PP2A function. Furthermore, our studies show that PI3Kγ phosphorylates the endogenous inhibitor of PP2A, I2PP2A which then robustly binds and inhibits PP2A activity impairing β2AR dephosphorylation. Consistently, CRISPR ablation of I2PP2A relieves this inhibition on PP2A leading to increased PP2A activity. This reduces accumulation of phosphorylated βARs despite the presence of PI3Kγ showing an underappreciated regulation of PP2A by insulin through non-canonical recruitment of PI3Kγ that impairs β2AR function.

Protein Phosphatase 2A Improves Cardiac Functional Response to Ischemia and Sepsis.

Reversible protein phosphorylation is a posttranslational modification of regulatory proteins involved in cardiac signaling pathways. Here, we focus on the role of protein phosphatase 2A (PP2A) for cardiac gene expression and stress response using a transgenic mouse model with cardiac myocyte-specific overexpression of the catalytic subunit of PP2A (PP2A-TG). Gene and protein expression were assessed under basal conditions by gene chip analysis and Western blotting. Some cardiac genes related to the cell metabolism and to protein phosphorylation such as kinases and phosphatases were altered in PP2A-TG compared to wild type mice (WT). As cardiac stressors, a lipopolysaccharide (LPS)-induced sepsis in vivo and a global cardiac ischemia in vitro (stop-flow isolated perfused heart model) were examined. Whereas the basal cardiac function was reduced in PP2A-TG as studied by echocardiography or as studied in the isolated work-performing heart, the acute LPS- or ischemia-induced cardiac dysfunction deteriorated less in PP2A-TG compared to WT. From the data, we conclude that increased PP2A activity may influence the acute stress tolerance of cardiac myocytes.

Chk1 Inhibition Ameliorates Alzheimer’s Disease Pathogenesis and Cognitive Dysfunction Through CIP2A/PP2A Signaling

Alzheimer’s disease (AD) is the most common neurodegenerative disease with limited therapeutic strategies. Cell cycle checkpoint protein kinase 1 (Chk1) is a Ser/Thr protein kinase which is activated in response to DNA damage, the latter which is an early event in AD. However, whether DNA damage-induced Chk1 activation participates in the development of AD and Chk1 inhibition ameliorates AD-like pathogenesis remain unclarified. Here, we demonstrate that Chk1 activity and the levels of protein phosphatase 2A (PP2A) inhibitory protein CIP2A are elevated in AD human brains, APP/PS1 transgenic mice, and primary neurons with Aβ treatment. Chk1 overexpression induces CIP2A upregulation, PP2A inhibition, tau and APP hyperphosphorylation, synaptic impairments, and cognitive memory deficit in mice. Moreover, Chk1 inhibitor (GDC0575) effectively increases PP2A activity, decreases tau phosphorylation, and inhibits Aβ overproduction in AD cell models. GDC0575 also reverses AD-like cognitive deficits and prevents neuron loss and synaptic impairments in APP/PS1 mice. In conclusion, our study uncovers a mechanism by which DNA damage-induced Chk1 activation promotes CIP2A-mediated tau and APP hyperphosphorylation and cognitive dysfunction in Alzheimer’s disease and highlights the therapeutic potential of Chk1 inhibitors in AD.

Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A): Could It Be a Promising Biomarker and Therapeutic Target in Parkinson\u2019s Disease?

Parkinson’s disease (PD) is an incurable neurodegenerative disease characterized by aggregation of pathological alpha-synuclein (α-syn) and loss of dopaminergic neuron in the substantia nigra. Inhibition of phosphorylation of the α-syn has been shown to mediate alleviation of PD-related pathology. Protein phosphatase 2A (PP2A), an important serine/threonine phosphatase, plays an essential role in catalyzing dephosphorylation of the α-syn. Here, we identified and validated cancerous inhibitor of PP2A (CIP2A), as a potential diagnostic biomarker for PD. Our data showed that plasma CIP2A concentrations in PD patients were significantly lower compared to age- and sex-matched controls, 1.721 (1.435–2.428) ng/ml vs 3.051(2.36–5.475) ng/ml, p < 0.0001. The area under the curve of the plasma CIP2A in distinguishing PD from the age- and sex-matched controls was 0.776. In addition, we evaluated the role of CIP2A in PD-related pathogenesis in PD cellular and MPTP-induced mouse model. The results demonstrated that CIP2A is upregulated in PD cellular and MPTP-induced mouse models. Besides, suppression of the CIP2A expression alleviates rotenone induced aggregation of the α-syn as well as phosphorylation of the α-syn in SH-SY5Y cells, which is associated with increased PP2A activity. Taken together, our data demonstrated that CIP2A plays an essential role in the mechanisms related to PD development and might be a novel PD biomarker.

Renal AT2 Receptors Mediate Natriuresis via Protein Phosphatase PP2A.

How signals from activated angiotensin type-2 receptors (AT2R) mediate inhibition of sodium ion (Na+) reabsorption in renal proximal tubule cells is currently unknown. Protein phosphatases including PP2A (protein phosphatase 2A) have been implicated in AT2R signaling in tissues other than kidney. We investigated whether inhibition of protein phosphatase PP2A reduced AT2R-mediated natriuresis and evaluated changes in PP2A activity and localization after renal AT2R activation in normal 4- and 10-week-old control Wistar-Kyoto rats and 4-week-old prehypertensive and 10-week-old hypertensive spontaneously hypertensive rats. In Wistar-Kyoto rats, direct renal interstitial administration of selective AT2R nonpeptide agonist Compound-21 (C-21) increased renal interstitial cyclic GMP (cGMP) levels, urine Na+ excretion, and simultaneously increased PP2A activity ≈2-fold in homogenates of renal cortical tubules. The cyclic GMP and natriuretic responses were abolished by concurrent renal interstitial administration of protein phosphatase inhibitor calyculin A. In renal proximal tubule cells in response to C-21, PP2A subunits A, B55α and C, but not B56γ, were recruited to apical plasma membranes together with AT2Rs. Calyculin A treatment abolished C-21-induced translocation of both AT2R and PP2A regulatory subunit B55α to apical plasma membranes. Immunoprecipitation of AT2R solubilized from renal cortical homogenates demonstrated physical association of AT2R with PP2A A, B55α, and C but not B56γ subunits. In contrast, in spontaneously hypertensive rats, administration of C-21 did not alter urine Na+ excretion or PP2A activity and failed to translocate AT2Rs and PP2A subunits to apical plasma membranes. In renal proximal tubule cells of Wistar-Kyoto rats, PP2A is activated and PP2A subunits AB55αC are recruited to C-21-activated AT2Rs during induction of natriuresis. This response is defective in prehypertensive and hypertensive spontaneously hypertensive rats, presenting a potential novel therapeutic target for treating renal Na+ retention and hypertension.

Metformin Increases Protein Phosphatase 2A Activity in Primary Human Skeletal Muscle Cells Derived from Lean Healthy Participants.

Objective To investigate if PP2A plays a role in metformin-induced insulin sensitivity improvement in human skeletal muscle cells. Participants. Eight lean insulin-sensitive nondiabetic participants (4 females and 4 males; age: 21.0 ± 1.0 years; BMI: 22.0 ± 0.7 kg/m2; 2-hour OGTT: 97.0 ± 6.0 mg/dl; HbA1c: 5.3 ± 0.1%; fasting plasma glucose: 87.0 ± 2.0 mg/dl; M value; 11.0 ± 1.0 mg/kgBW/min). Design A hyperinsulinemic-euglycemic clamp was performed to assess insulin sensitivity in human subjects, and skeletal muscle biopsy samples were obtained. Primary human skeletal muscle cells (shown to retain metabolic characteristics of donors) were cultured from these muscle biopsies that included 8 lean insulin-sensitive participants. Cultured cells were expanded, differentiated into myotubes, and treated with 50 μM metformin for 24 hours before harvesting. PP2Ac activity was measured by a phosphatase activity assay kit (Millipore) according to the manufacturer's protocol. Results The results indicated that metformin significantly increased the activity of PP2A in the myotubes for all 8 lean insulin-sensitive nondiabetic participants, and the average fold increase is 1.54 ± 0.11 (P < 0.001). Conclusions These results provided the first evidence that metformin can activate PP2A in human skeletal muscle cells derived from lean healthy insulin-sensitive participants and may help to understand metformin's action in skeletal muscle in humans.

Consuming oxidative frying oil impairs cardiac energy production and calcium recycling, causing cardiac hypertrophy, fibrosis and diastolic dysfunction in male Sprague Dawley rats

With regards to cardiovascular health, frequent consumption of fried foods is discouraged, despite a lack of clear evidence of a direct link between eating oxidative frying oil (OFO) and cardiovascular diseases. In this study, male Sprague Dawley rats were exposed to diets containing fresh or fried soybean oil (groups C and O, respectively) from in utero to 28 weeks of age. A subset of rats in group O was supplemented with vitamin E (500 mg/kg of DL-α-tocopherol acetate; group OE) from 8 week of age onward to mitigate oxidative stress associated with OFO ingestion. Echocardiography, cardiac histology and indices associated with ATP production and calcium cycling in cardiac tissues were measured. Compared to group C, there was cardiac hypertrophy, fibrosis and diastolic dysfunction, in groups O and OE, with no differences between the latter two groups. Although cardiac mRNA levels of genes associated with mitochondrial biogenesis and function were increased, there were lower ATP concentrations and higher transcripts of uncoupling proteins in groups O and OE than in group C. In addition, decreases in phosphorylation of phospholamban and Ca2+/calmodulin-dependent protein kinase II activity, plus increased protein phosphatase 2A activity in groups O and OE, implied calcium cycling required for cardiac function was disrupted by OFO consumption. We concluded that long-term OFO exposure resulted in cardiac hypertrophy, fibrosis and diastolic dysfunction that was not mitigated by vitamin E supplementation. Underlying mechanisms were partly attributed to inefficient energy production via uncoupled phosphorylation and disrupted calcium cycling.

Inhibiting protein phosphatase 2A increases the antitumor effect of protein arginine methyltransferase 5 inhibition in models of glioblastoma.

Despite multi-model therapy of maximal surgical resection, radiation, chemotherapy, and tumor-treating fields, the median survival of glioblastoma (GBM) patients is less than 15 months. Protein arginine methyltransferase 5 (PRMT5) catalyzes the symmetric dimethylation of arginine residues and is overexpressed in GBM. Inhibition of PRMT5 causes senescence in stem-like GBM tumor cells. LB100, a first-in-class small molecular inhibitor of protein phosphatase 2A (PP2A), can sensitize therapy-resistant tumor cells. Here, we tested the anti-GBM effect of concurrent PRMT5 and PP2A inhibition. Patient-derived primary GBM neurospheres (GBMNS), transfected with PRMT5 target-specific siRNA, were treated with LB100 and subjected to in vitro assays including PP2A activity and western blot. The intracranial mouse xenograft model was used to test the in vivo antitumor efficacy of combination treatment. We found that PRMT5 depletion increased PP2A activity in GBMNS. LB100 treatment significantly reduced the viability of PRMT5-depleted GBMNS compared to PRMT5-intact GBMNS. LB100 enhanced G1 cell cycle arrest induced by PRMT5 depletion. Combination therapy also increased the expression of phospho-MLKL. Necrostatin-1 rescued PRMT5-depleted cells from the cytotoxic effects of LB100, indicating that necroptosis caused the enhanced cytotoxicity of combination therapy. In the in vivo mouse tumor xenograft model, LB100 treatment combined with transient depletion of PRMT5 significantly decreased tumor size and prolonged survival, while LB100 treatment alone had no survival benefit. Overall, combined PRMT5 and PP2A inhibition had significantly greater antitumor effects than PRMT5 inhibition alone.

STEM-23. INHIBITING PROTEIN PHOSPHATASE 2A INCREASES THE ANTITUMOR EFFECT OF PROTEIN ARGININE METHYLTRANSFERASE 5 INHIBITION IN MODELS OF GLIOBLASTOMA

Abstract BACKGROUND: Despite multi-model therapy of maximal surgical resection, radiation, chemotherapy, and tumor treating fields, median survival of Glioblastoma (GBM) patients is less than two years. Protein Arginine Methyltransferase 5 (PRMT5) catalyzes the symmetric di-methylation of arginine residues and is overexpressed in GBM. Inhibition of PRMT5 causes senescence of immature GBM tumor cells. LB100, first-in-class small molecule inhibitor of Protein Phosphatase 2A (PP2A) can sensitize therapy-resistant tumor cells. Here, we tested the anti-GBM effect of concurrent PRMT5 and PP2A inhibition. Methods: Patient-derived primary GBM neurospheres (GBMNS), transfected with PRMT5 target-specific siRNA were treated with LB100 and subjected to in vitro assays including PP2A activity and western blot. The intracranial mouse xenograft model was used to test the in vivo antitumor efficacy of combination treatment. Results: We found that PRMT5-depletion increased PP2A activity in GBMNS. LB100 treatment significantly reduced the viability of PRMT5-depleted GBMNS compared to PRMT5 intact GBMNS. LB100 enhanced the G1 cell cycle arrest induced by PRMT5-depletion. Combination therapy also increased the expression of phospho-MLKL. Necrostatin-1 rescued PRMT5-depleted cells from the cytotoxic effects of LB100, indicating that necroptosis caused the enhanced cytotoxicity of combination therapy. In the in vivo mouse tumor xenograft model, LB100 treatment combined with transient depletion of PRMT5 significantly decreased tumor size and prolonged mice survival, while LB100 treatment alone had no survival benefit. Conclusion: Overall, combined PRMT5 and PP2A inhibition had significantly greater antitumor effects than PRMT5 inhibition alone.

Abstract 425: Protein Kinase Activity of Pi3kγ Regulates Insulin-βArs Cross-talk

β adrenergic receptor (βAR) function is regulated by G-protein coupled receptor (GPCR) kinase (GRK) driven desensitization and protein phosphatase 2A (PP2A) mediated resensitization to its classical agonist. Surprisingly, Insulin modulates (βARs) function thus regulating cardiac function. Although insulin is known to modulate βAR function through GRKs, less is known about insulin mediated resensitization mechanisms. PI3Kγ is activated by GPCRs regulates resensitization by inhibiting PP2A activity. Therefore, we tested whether insulin could mediate βAR dysfunction through inhibition of resensitization. Co-immunoprecipitation in cardiac lysates and surface plasmon resonance (SPR) with purified proteins show that PI3Kγ interacts with GRK-2 resulting in recruitment of GRK2-PI3Kγ to the β2AR complex upon insulin. Furthermore, use of PI3K inhibitors significantly reduced Insulin-stimulated β2ARs phosphorylation in HEK293 cells. The role of PI3Kγ was further validated by the loss of insulin stimulated β 2 AR phosphorylation in PI3Kγ-knockout (KO) mouse embryonic fibroblasts (MEFs), shRNA and CRIPSR knockdown of PI3Kγ. Data shows that PI3Kγ inhibits PP2A activity at the βAR complex upon insulin while loss of PI3Kγ unravels this inhibition resulting in increased PP2A activity leading to β2AR dephosphorylation and resensitization. Mechanistically, PI3Kγ inhibits PP2A activity at the β2AR complex by phosphorylating an endogenous inhibitor of PP2A (I2PP2A). CRISPR knockout and siRNA knockdown of endogenous I2PP2A in HEK293 cells restored PP2A activity resulting in β2AR dephosphorylation despite PI3Kγ. Furthermore, β blocker (propranolol) pretreatment did not affect β2AR phosphorylation and there was no β-arrestin recruitment to the βAR complex with Insulin. Together these studies show that Insulin mediates β2AR desensitization through β-agonist and β-arrestin independent mechanisms wherein, PI3Kγ-mediated regulation of PP2A activity plays a pivotal role in cardiac βAR function in hyperinsulemic conditions like diabetes and obesity.

1883-P: Metformin Increases Protein Phosphatase 2A Activity in Primary Skeletal Muscle Cells Derived from Lean Healthy Insulin-Sensitive Human Participants

Skeletal muscle insulin resistance is one of the primary contributors of type 2 diabetes (T2D). Metformin is the first-line drug for the treatment of T2D. Main known effects of metformin include inhibition of glucose production in liver and attenuation of insulin resistance in skeletal muscle. However, molecular mechanism of metformin’s action in skeletal muscle is not well understood. Protein phosphatase 2A (PP2A), a major serine/threonine phosphatase, plays a pivotal role in cellular processes (e.g., signal transduction, apoptosis), and acts through dephosphorylating key signaling molecules such as AKT, AMPK, etc. However, whether or not PP2A plays a role in metformin-induced insulin sensitivity improvement in human skeletal muscle cells remains to be elucidated. In the present study, hyperinsulinemic-euglycemic clamp was performed to assess insulin sensitivity in human subjects and skeletal muscle biopsy samples were obtained. Primary human skeletal muscle cells (shown to retain metabolic characteristics of donors) were cultured from these muscle biopsies from 8 lean insulin sensitive participants (4 Female and 4 male; age: 21.4±0.8 yrs; BMI: 22.1±0.7 kg/m2; 2-hour OGTT: 96.5± 6.3 mg/dl; HbA1c: 5.3±0.1 %; fasting plasma glucose: 87.5±1.4 mg/dl; M-value; 10.9±0.8 mg/kgBW/min). The cells were expanded, differentiated into myotubes, and treated with 50µM metformin (the physiologic concentration) for 24 hours before harvesting. PP2A activity was measured by a phosphatase activity assay (Millipore). The results indicated that metformin increased the activity of PP2A in the myotubes derived from all 8 participants, and the average fold increase is 1.59±0.13 (p&amp;lt;0.05). These results provided the first evidence to suggest that metformin promotes activation of PP2A in human skeletal muscle cells derived from lean insulin sensitive participants, and may help to understand metformin’s action in skeletal muscle in humans. Disclosure A.H. Mestareehi: None. B. Seyoum: None. Z. Msallaty: None. A.P. Mallisho: None. X. Zhang: None. A. Kowluru: None. Z. Yi: None. Funding American Diabetes Association (1-13-TS-27 to Z.Y.); National Institutes of Health (R01DK107666, R01DK081750 to Z.Y.), (T32HL120822 to A.H.M.); DOTS Scholars (to A.H.M., Z.Y.)

Role of PP2A in Racial Differences in Endothelial Dysfunction

BackgroundAfrican Americans (AA) exhibit a higher prevalence of endothelial dysfunction and an exaggerated response to inflammatory cytokines compared to Caucasian Americans (CA). Endothelial dysfunction is characterized by a reduction in endothelial nitric oxide synthase (eNOS) activity and ultimately a lower nitric oxide production. Data obtained from bovine endothelial cells show that a reduction in eNOS activity in response to an inflammatory stimulus is mediated by protein phosphatase 2A (PP2A). Additionally, higher PP2A activity is associated with a decrease in the phosphorylation of PP2A at the Tyrosine 307 residue (p‐PP2AY307). Therefore, the purpose of this study was to investigate the role of PP2A in racial differences seen in endothelial dysfunction in response to an inflammatory stimulus.MethodsHuman umbilical vein endothelial cells (HUVECs) (n=6 cell lines; 3 CA &amp; 3 AA) were cultured under two conditions: control or tumor necrosis factor‐α (TNF‐α) (10 ng/mL, 4 hours). Western blotting was used to measure protein expression of p‐PP2AY307, PP2A, p‐eNOSS1177 and eNOS. A two‐way ANOVA was used to assess the effects of race and conditions.ResultsNo racial differences were found in the protein expression of PP2A (Fig. 1B) and eNOS (Fig. 2B) at baseline or with TNF‐α stimulation. However, AA had a lower p‐PP2AY307 expression (p=0.021; Fig. 1A) compared to CA HUVECs under both conditions. Additionally, AA had a lower p‐eNOSS1177 expression (p=0.008; Fig. 2A) compared to CA HUVECs under both condition.ConclusionLower p‐PP2AY307 and p‐eNOSS1177 levels in AA endothelial cells could be attributed to an increase in PP2A activity that can ultimately inhibit eNOS phosphorylation and decrease nitic oxide production. Therefore, PP2A activity can explain, in part, racial differences observed in endothelial dysfunction and consequently cardiovascular diseases.Support or Funding InformationRacial differences in p‐PP2AY307 and PP2A protein expression in HUVECs after 4 hours of incubation with TNF‐α (10ng/ml).A) Densiometric quantification and representative Western blot of p‐PP2AY307 and B) Densiometric quantification and representative Western blot of PP2A. (# p= .02 in AA compared to CA HUVECs, n= 4 independent experiments. Data are shown as mean ± SEM).Figure 1Racial differences in p‐eNOSS1177 and eNOS protein expression in HUVECs after 4 hours of incubation with TNF‐α (10ng/ml).A) Densiometric quantification and representative Western blot of p‐eNOSS1177 and B) Densiometric quantification and representative Western blot of eNOS. (# p= .008 in AA compared to CA HUVECs, n= 4 independent experiments. Data are shown as mean ± SEM).Figure 2AcknowledgementsFunded partially by AU PGOP Fellowship.

Serine/threonine phosphatase PP2A is essential for optimal B cell function.

Protein phosphatase 2A (PP2A), a serine/threonine phosphatase, has been shown to control T cell function. We found that in vitro-activated B cells and B cells from various lupus-prone mice and patients with systemic lupus erythematosus display increased PP2A activity. To understand the contribution of PP2A to B cell function, we generated a Cd19CrePpp2r1afl/fl (flox/flox) mouse which lacks functional PP2A only in B cells. Flox/flox mice displayed reduced spontaneous germinal center formation and decreased responses to T cell-dependent and T-independent antigens, while their B cells responded poorly in vitro to stimulation with an anti-CD40 antibody or CpG in the presence of IL-4. Transcriptome and metabolome studies revealed altered nicotinamide adenine dinucleotide (NAD) and purine/pyrimidine metabolism and increased expression of purine nucleoside phosphorylase in PP2A-deficient B cells. Our results demonstrate that PP2A is required for optimal B cell function and may contribute to increased B cell activity in systemic autoimmunity.

Direct Activation of Protein Phosphatase 2A (PP2A) by Tricyclic Sulfonamides Ameliorates Alzheimer's Disease Pathogenesis in Cell and Animal Models.

Alzheimer's disease (AD) is a multifactorial neurodegenerative disease for which there are limited therapeutic strategies. Protein phosphatase 2A (PP2A) activity is decreased in AD brains, which promotes the hyperphosphorylation of Tau and APP, thus participate in the formation of neurofibrillary tangles (NFTs) and β-amyloid (Aβ) overproduction. In this study, the effect of synthetic tricyclic sulfonamide PP2A activators (aka SMAPs) on reducing AD-like pathogenesis was evaluated in AD cell models and AD-like hyperhomocysteinemia (HHcy) rat models. SMAPs effectively increased PP2A activity, and decreased tau phosphorylation and Aβ40/42 levels in AD cell models. In HHcy-AD rat models, cognitive impairments induced by HHcy were rescued by SMAP administration. HHcy-induced tau hyperphosphorylation and Aβ overproduction were ameliorated through increasing PP2A activity on compound treatment. Importantly, SMAP therapy also prevented neuronal cell spine loss and neuronal synapse impairment in the hippocampus of HHcy-AD rats. In summary, our data reveal that pharmacological PP2A reactivation may be a novel therapeutic strategy for AD treatment, and that the tricyclic sulfonamides constitute a novel candidate class of AD therapeutic.

Prolyl oligopeptidase inhibition activates autophagy via protein phosphatase 2A

Prolyl oligopeptidase (PREP) is a serine protease that has been studied particularly in the context of neurodegenerative diseases for decades but its physiological function has remained unclear. We have previously found that PREP negatively regulates beclin1-mediated macroautophagy (autophagy), and that PREP inhibition by a small-molecule inhibitor induces clearance of protein aggregates in Parkinson’s disease models. Since autophagy induction has been suggested as a potential therapy for several diseases, we wanted to further characterize how PREP regulates autophagy. We measured the levels of various kinases and proteins regulating beclin1-autophagy in HEK-293 and SH-SY5Y cell cultures after PREP inhibition, PREP deletion, and PREP overexpression and restoration, and verified the results in vivo by using PREP knock-out and wild-type mouse tissue where PREP was restored or overexpressed, respectively. We found that PREP regulates autophagy by interacting with protein phosphatase 2A (PP2A) and its endogenous inhibitor, protein phosphatase methylesterase 1 (PME1), and activator (protein phosphatase 2 phosphatase activator, PTPA), thus adjusting its activity and the levels of PP2A in the intracellular pool. PREP inhibition and deletion increased PP2A activity, leading to activation of death-associated protein kinase 1 (DAPK1), beclin1 phosphorylation and induced autophagy while PREP overexpression reduced this. Lowered activity of PP2A is connected to several neurodegenerative disorders and cancers, and PP2A activators would have enormous potential as drug therapy but development of such compounds has been a challenge. The concept of PREP inhibition has been proved safe, and therefore, our study supports the further development of PREP inhibitors as PP2A activators.

Protein phosphatase 2A controls ongoing DNA replication by binding to and regulating cell division cycle 45 (CDC45)

Genomic replication is a highly regulated process and represents both a potential benefit and liability to rapidly dividing cells; however, the precise post-translational mechanisms regulating genomic replication are incompletely understood. Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase that regulates a diverse array of cellular processes. Here, utilizing both a gain-of-function chemical biology approach and loss-of-function genetic approaches to modulate PP2A activity, we found that PP2A regulates DNA replication. We demonstrate that increased PP2A activity can interrupt ongoing DNA replication, resulting in a prolonged S phase. The impaired replication resulted in a collapse of replication forks, inducing dsDNA breaks, homologous recombination, and a PP2A-dependent replication stress response. Additionally, we show that during replication, PP2A exists in complex with cell division cycle 45 (CDC45) and that increased PP2A activity caused dissociation of CDC45 and polymerase α from the replisome. Furthermore, we found that individuals harboring mutations in the PP2A Aα gene have a higher fraction of genomic alterations, suggesting that PP2A regulates ongoing replication as a mechanism for maintaining genomic integrity. These results reveal a new function for PP2A in regulating ongoing DNA replication and a potential role for PP2A in the intra-S-phase checkpoint.

Metformin prevents the pathological browning of subcutaneous white adipose tissue

ObjectiveBrowning, the conversion of white adipose tissue (WAT) to a beige phenotype, has gained interest as a strategy to induce weight loss and improve insulin resistance in metabolic disorders. However, for hypermetabolic conditions stemming from burn trauma or cancer cachexia, browning is thought to contribute to energy wasting and supraphysiological nutritional requirements. Metformin's impact on this phenomenon and underlying mechanisms have not been explored. MethodsWe used both a murine burn model and human ex vivo adipose explants to assess metformin and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR)'s effects on the development of subcutaneous beige adipose. Enzymes involved in fat homeostasis and browning, as well as mitochondrial dynamics, were assessed to determine metformin's effects. ResultsTreatment with the biguanide metformin lowers lipolysis in beige fat by inducing protein phosphatase 2A (PP2A) independently of adenosine monophosphate kinase (AMPK) activation. Increased PP2A activity catalyzes the dephosphorylation of acetyl-CoA carboxylase (Ser 79) and hormone sensitive lipase (Ser 660), thus promoting fat storage and the “whitening” of otherwise lipolytic beige adipocytes. Moreover, co-incubation of metformin with the PP2A inhibitor okadaic acid countered the anti-lipolytic effects of this biguanide in human adipose. Additionally, we show that metformin does not activate this pathway in the WAT of control mice and that AICAR sustains the browning of white adipose, offering further evidence that metformin acts independently of this cellular energy sensor. ConclusionsThis work provides novel insights into the mechanistic underpinnings of metformin's therapeutic benefits and potential as an agent to reduce the lipotoxicity associated with hypermetabolism and adipose browning.

Targeting SET to restore PP2A activity disrupts an oncogenic CIP2A-feedforward loop and impairs triple negative breast cancer progression.

BackgroundTriple-negative breast cancer (TNBC) remains difficult to be targeted. SET and cancerous inhibitor of protein phosphatase 2A (CIP2A) are intrinsic protein-interacting inhibitors of protein phosphatase 2A (PP2A) and frequently overexpressed in cancers, whereas reactivating PP2A activity has been postulated as an anti-cancer strategy. Here we explored this strategy in TNBC.MethodsData from The Cancer Genome Atlas (TCGA) database was analyzed. TNBC cell lines were used for in vitro studies. Cell viability was examined by MTT assay. The apoptotic cells were examined by flow cytometry and Western blot. A SET-PP2A protein-protein interaction antagonist TD19 was used to disrupt signal transduction. In vivo efficacy of TD19 was tested in MDA-MB-468-xenografted animal model.FindingsTCGA data revealed upregulation of SET and CIP2A and positive correlation of these two gene expressions in TNBC tumors. Ectopic SET or CIP2A increased cell viability, migration, and invasion of TNBC cells. Notably ERK inhibition increased PP2A activity. ERK activation is known crucial for Elk-1 activity, a transcriptional factor regulating CIP2A expression, we hypothesized an oncogenic feedforward loop consisting of pERK/pElk-1/CIP2A/PP2A. This loop was validated by knockdown of PP2A and ectopic expression of Elk-1, showing reciprocal changes in loop members. In addition, ectopic expression of SET increased pAkt, pERK, pElk-1 and CIP2A expressions, suggesting a positive linkage between SET and CIP2A signaling. Moreover, TD19 disrupted this CIP2A-feedforward loop by restoring PP2A activity, demonstrating in vitro and in vivo anti-cancer activity. Mechanistically, TD19 downregulated CIP2A mRNA via inhibiting pERK-mediated Elk-1 nuclear translocation thereby decreased Elk-1 binding to the CIP2A promoter.InterpretationThese findings suggested that a novel oncogenic CIP2A-feedforward loop contributes to TNBC progression and targeting SET to disrupt this oncogenic CIP2A loop showed therapeutic potential in TNBC.