PP2A Activity Research Articles

Abstract 5190: Oncoprotein SET determines the radiosensitivity of hepatocellular carcinoma cell and antagonizing SET augments the effects of radiotherapy via reactivating PP2A-mediated Akt downregulation

Abstract Background: SET is a novel oncoprotein with growing evidence suggesting its roles in promoting the survival of hepatocellular carcinoma (HCC) and other types of malignant disease. Notably, pre-clinical data suggesting SET overexpression in cancer cell leaded to the development of resistance to various anti-cancer medications, and antagonizing SET reversely enhanced therapeutic efficacy. Radiotherapy (RT) is a commonly used anti-cancer modality, but its role for the treatment of HCC is largely limited for the concern of endogenous radioresistance. In this report, we explored the role of SET regarding the radiosensitivity of HCC cells. Furthermore, we explored whether SET serve as a valid target to enhance RT against HCC. Method: A panel of HCC cell lines, including Hep3B, PLC5, HA22T and HA59T, were used for in vitro experiments, and the PLC5 subcutaneous xenograft mice model was used for in vivo testing. HCC cells and tumors were treated with RT and/or SET antagonist and harvested for subsequent experiments. The viability of cancer cells were assessed by sub-G1, colony and sphere formation assay. Molecular events were determined by western blot and PP2A activity. Result: Through manipulating the expression of SET in cancer cells, we showed the crucial role of SET in mediating the effects of RT in HCC cells. Using colony formation and hepatosphere formation assay, we showed that RT significantly reduced the number and size of both tumor colony and sphere formed by Hep3B and PLC5 cells. In contrast, ectopic expression of SET in Hep3B and PLC5 cells abolished the effects of RT. More importantly, SET-knockdown oppositely potentiated the RT-induced growth inhibition in HCC cells. Based on our findings, we hypothesized that targeting SET may exert potential to enhance RT for the treatment of HCC. In order to validate our hypothesis, we tested the effects of RT in combine with a novel SET antagonist, EMQA, developed and characterized in our previous works. As expected, we found that EMQA significantly potentiated the anti-HCC effects of RT in vitro and in vivo. Moreover, we found that antagonizing SET to restore PP2A-mediated p-Akt downregulation was responsible for the synergism between EMQA and RT. Conclusion Oncoprotein SET plays a critical role in affecting the radiosensitivity of HCC cells. Using SET antagonist plus RT showed promising results in pre-clinical HCC models, and yields further investigation. Citation Format: Man-Hsin Hung, Chao-Yuan Huang, Chih-Ting Shih, Min-Hsien Tsai, Yung-Jen Hsiao, Tzu-I Chao, Feng-Shu Hsieh, Chung-Wai Shiau, Kuen-Feng Chen. Oncoprotein SET determines the radiosensitivity of hepatocellular carcinoma cell and antagonizing SET augments the effects of radiotherapy via reactivating PP2A-mediated Akt downregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5190. doi:10.1158/1538-7445.AM2017-5190

Abstract 2382: RACK1 regulates PKA activity to drive tumorigenesis in colon cancer

Abstract RACK1 (Receptor for Activated C Kinases) is a scaffolding protein with 7 WD repeats that interacts with the Insulin-like Growth Factor I receptor (IGF-IR), integrins, and other signalling proteins. RACK1 functions as a scaffolding protein and regulator of many key biological processes and it is highly expressed in most tissue. We recently reported that RACK1 is essential for IGF-I-mediated regulation of PP2A activity and AGAP2 activity at focal adhesions. Downstream of IGF-1R signaling in cancer cells, the scaffolding properties of RACK1 are altered providing distinct migratory advantages to the cell and suggesting that RACK1 is an important regulator of IGF-I signalling in cancer progression. It is important to further characterise the role of RACK1 in cancer and to delineate the molecular mechanisms by which it regulates key signalling pathways. We have identified Protein Kinase A (PKA) as RACK1 interacting protein. PKA is a complex, multicomponent enzyme which is a fundamental protein that functions in cell survival, proliferation, and cytoskeletal remodelling among others. Our hypothesis is that RACK1 regulates the PKA axis in cancer cells leading to Akt and MAP Kinase activation downstream of IGF-1R signalling. We have demonstrated that RACK1 is required to mediate PKA activity in colon cancer cells. Although we show that PKA expression is not altered in colon cancer tissue, we believe that PKA activity is altered significantly in the disease state. Using PKA inhibitors, we have shown that PKA regulates IGF-I-mediated MAP Kinase activation in colon cancer cell lines. In conclusion, this research project will help elucidate the role of PKA in colon cancer. Inhibiting or promoting specific protein interactions with RACK1 will provide very novel therapeutic opportunities and anti-cancer drug targets. Citation Format: Sheri L. Hayes, Catríona M. Dowling, John C. Coffey, Patrick A. Kiely. RACK1 regulates PKA activity to drive tumorigenesis in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2382. doi:10.1158/1538-7445.AM2017-2382

Abstract 533: XIAP BIR domain suppresses miR-200a expression and subsequently promotes EGFR protein translation and anchorage-independent growth of bladder cancer cell

Abstract The X-linked inhibitor of apoptosis protein (XIAP) is a well-known potent apoptosis suppressor, and also participates in cancer cell biological behaviors, therefore attracting great attentions as a potential antineoplastic therapeutic target for past years. We found here that lacking of XIAP expression resulted in a remarkable suppression of EGFR expression, consequently leading to the deficient of anchorage-independent cell growth. Further study demonstrated that BIR domain of XIAP was crucial for regulating the EGFR translation by suppressing the transcription and expression of miR-200a. Mechanistic studies indicated that BIR domain activated the PP2A activity by decreasing the phosphorylation of PP2A at Tyr307 in its catalytic subunit, PP2A-C. Such activated PP2A prevented the deviant phosphorylation and activation of MAPK kinases/MAPKs, their downstream effector c-Jun, and in turn inhibiting transcription of c-Jun-regulated the miR-200a. Collectively, our study uncovered a novel function of BIR domain of XIAP in regulating the EGFR translation, providing significant insight into the understanding of the XIAP overexpression in the cancer development and progression, further offering a new theoretical support for using XIAP BIR domain and EGFR as targets for cancer therapy. Citation Format: Xin Liao, Chao Huang, Jingxia Li, Chuanshu Huang. XIAP BIR domain suppresses miR-200a expression and subsequently promotes EGFR protein translation and anchorage-independent growth of bladder cancer cell [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 533. doi:10.1158/1538-7445.AM2017-533

Fyn kinase mediates cortical actin ring depolymerization required for mast cell migration in response to TGF-β in mice.

Transforming growth factor-β (TGF-β) is a potent mast cell (MC) chemoattractant able to modulate local inflammatory reactions. The molecular mechanism leading to TGF-β-directed MC migration is not fully described. Here we analyzed the role of the Src family protein kinase Fyn on the main TGF-β-induced cytoskeletal changes leading to MC migration. Utilizing bone marrow-derived mast cells (BMMCs) from WT and Fyn-deficient mice we found that BMMC migration to TGF-β was impaired in the absence of the kinase. TGF-β caused depolymerization of the cortical actin ring and changes on the phosphorylation of cofilin, LIMK and CAMKII only in WT cells. Defective cofilin activation and phosphorylation of regulatory proteins was detected in Fyn-deficient BMMCs and this finding correlated with a lower activity of the catalytic subunit of the phosphatase PP2A. Diminished TGF-β-induced chemotaxis of Fyn-deficient cells was also observed in an in vivo model of MC migration (bleomycin-induced scleroderma). Our results show that Fyn kinase is an important positive effector of TGF-β-induced chemotaxis through the control of PP2A activity and this is relevant to pathological processes that are related to TGF-β-dependent mast cell migration.

PP2A Controls Genome Integrity by Integrating Nutrient-Sensing and Metabolic Pathways with the DNA Damage Response

SummaryMec1ATR mediates the DNA damage response (DDR), integrating chromosomal signals and mechanical stimuli. We show that the PP2A phosphatases, ceramide-activated enzymes, couple cell metabolism with the DDR. Using genomic screens, metabolic analysis, and genetic and pharmacological studies, we found that PP2A attenuates the DDR and that three metabolic circuits influence the DDR by modulating PP2A activity. Irc21, a putative cytochrome b5 reductase that promotes the condensation reaction generating dihydroceramides (DHCs), and Ppm1, a PP2A methyltransferase, counteract the DDR by activating PP2A; conversely, the nutrient-sensing TORC1-Tap42 axis sustains DDR activation by inhibiting PP2A. Loss-of-function mutations in IRC21, PPM1, and PP2A and hyperactive tap42 alleles rescue mec1 mutants. Ceramides synergize with rapamycin, a TORC1 inhibitor, in counteracting the DDR. Hence, PP2A integrates nutrient-sensing and metabolic pathways to attenuate the Mec1ATR response. Our observations imply that metabolic changes affect genome integrity and may help with exploiting therapeutic options and repositioning known drugs.

Targeted activation of the SHP-1/PP2A signaling axis elicits apoptosis of chronic lymphocytic leukemia cells.

Lyn, a member of the Src family of kinases, is a key factor in the dysregulation of survival and apoptotic pathways of malignant B cells in chronic lymphocytic leukemia. One of the effects of Lyn’s action is spatial and functional segregation of the tyrosine phosphatase SHP-1 into two pools, one beneath the plasma membrane in an active state promoting pro-survival signals, the other in the cytosol in an inhibited conformation and unable to counter the elevated level of cytosolic tyrosine phosphorylation. We herein show that SHP-1 activity can be elicited directly by nintedanib, an agent also known as a triple angiokinase inhibitor, circumventing the phospho-S591-dependent inhibition of the phosphatase, leading to the dephosphorylation of pro-apoptotic players such as procaspase-8 and serine/threonine phosphatase 2A, eventually triggering apoptosis. Furthermore, the activation of PP2A by using MP07-66, a novel FTY720 analog, stimulated SHP-1 activity via dephosphorylation of phospho-S591, which unveiled the existence of a positive feedback signaling loop involving the two phosphatases. In addition to providing further insights into the molecular basis of this disease, our findings indicate that the PP2A/SHP-1 axis may emerge as an attractive, novel target for the development of alternative strategies in the treatment of chronic lymphocytic leukemia.

17β-Estradiol and/or estrogen receptor alpha blocks isoproterenol-induced calcium accumulation and hypertrophy via GSK3β/PP2A/NFAT3/ANP pathway.

The present study was aimed to investigate the protective effects of 17β-estradiol (E2) and estrogen receptor α (ERα) on isoproterenol (ISO)-treated H9c2 cardiomyoblast cells. In the present study, we treated H9c2 cells with ISO, a β-adrenergic receptor agonist, to induce myocardiac hypertrophy. Pre-administration of E2 or ERα (induced by doxycycline) and E2 plus ERα significantly prevented ISO-induced increase of cell size and cytosolic calcium accumulation, accompanied with increased mRNA of atrial natriuretic peptide and brain natriuretic peptide. However, ICI-ERs antagonist, and melatonin, a specific inhibitor for ERα, reversed the cardioprotective effects, suggesting that E2 action was mediated through ERα. Further evidences showed that E2 and ERα increased the protein level of GSK3β and protein phosphatase 2a inhibitor 2 (I2-PP2A), which subsequently enhanced the activation of I2-PP2A by disrupting PP2A activity and maintains normal calcium outflow. Collectively, E2 and ERα inhibited hypertrophy by preventing cytosol calcium accumulation and by inhibiting the association between PP2A with Na+-Ca2+ exchanger via GSK3β and I2-PP2A activation.

Zanthoxylum avicennae extract enhances GSK-3β to attenuate β-catenin via phosphatase 2A to block metastatic effects of HA22T cells and hepatocellular carcinoma xenografted nude mice.

Hepatocellular carcinoma (HCC) metastasis is often associated with the activation of Wnt/β-catenin signaling pathway. Zanthoxylum avicennae (Ying Bu Bo, YBB), a traditional herb with hepatoprotective effect, has been proven to inhibit human HCC in in vivo models however, the in vitro and in vivo effect of YBB on tumor metastasis is not clear yet. To determine whether YBB could inhibit HA22T human HCC cell by acting on β-catenin metastatic signaling in vitro and in vivo, HA22T cells were treated with different concentrations of YBB extracts (YBBE) and analyzed by Immunofluorescence staining assay, western blot analysis, siRNA mediated gene knock-down assays and co-immunoprecipitation assay. Additionally, the HA22T-implanted xenograft nude mice were used to confirm the assessed cellular effects. Mice treated with YBBEs showed a strong increasing trend in PP2Acα, GSK-3β, APC, and β-TrCP/HOS levels, however the expression of β-catenin, p-GSK-3β, TBX 3, and IL8 proteins showed a decreasing trend. YBBE significantly downregulated the nuclear and cytosolic β-catenin levels by facilitating the proteosomal degradation of β-catenin. Moreover, as observed by co-immunoprecipitation assay, YBBE directly promoted the protein interactions between GSK-3β, β-TrCP, APC, PP2A, and β-catenin. In conclusion, both in vitro and in vivo models clearly demonstrated that YBBE inhibits β-catenin involved metastatic signaling in highly metastatic HA22T cells through PP2A activation.

NNMT Silencing Activates Tumor Suppressor PP2A, Inactivates Oncogenic STKs, and Inhibits Tumor Forming Ability.

Purpose: To identify potential molecular hubs that regulate oncogenic kinases and target them to improve treatment outcomes for glioblastoma patients.Experimental Design: Data mining of The Cancer Genome Atlas datasets identified nicotinamide-N-methyl transferase (NNMT) as a prognostic marker for glioblastoma, an enzyme linked to the reorganization of the methylome. We tested our hypothesis that NNMT plays a crucial role by modulating protein methylation, leading to inactivation of tumor suppressors and activation of oncogenes. Further experiments were performed to understand the underlying biochemical mechanisms using glioblastoma patient samples, established, primary, and isogenic cells.Results: We demonstrate that NNMT outcompetes leucine carboxyl methyl transferase 1 (LCMT1) for methyl transfer from principal methyl donor SAM in biological systems. Inhibiting NNMT increased the availability of methyl groups for LCMT1 to methylate PP2A, resulting in the inhibition of oncogenic serine/threonine kinases (STK). Further, NNMT inhibition retained the radiosensitizer nicotinamide and enhanced radiation sensitivity. We have provided the biochemical rationale of how NNMT plays a vital role in inhibiting tumor suppressor PP2A while concomitantly activating STKs.Conclusions: We report the intricate novel mechanism in which NNMT inhibits tumor suppressor PP2A by reorganizing the methylome both at epigenome and proteome levels and concomitantly activating prosurvival STKs. In glioblastoma tumors with NNMT expression, activation of PP2A can be accomplished by FDA approved perphenazine (PPZ), which is currently used to treat mood disorders such as schizophrenia, bipolar disorder, etc. This study forms a foundation for further glioblastoma clinical trials using PPZ with standard of care treatment. Clin Cancer Res; 23(9); 2325-34. ©2016 AACR.

Peripheral Lipopolysaccharide Challenge Induces Long-Term Changes in Tyrosine Hydroxylase Regulation in the Adrenal Medulla.

Immune activation can alter the activity of adrenal chromaffin cells. The effect of immune activation by lipopolysaccharide (LPS) on the regulation of tyrosine hydroxylase (TH) in the adrenal medulla in vivo was determined between 1 day and 6 months after LPS injection. The plasma levels of eleven cytokines were reduced 1 day after LPS injection, whereas the level for interleukin-10 was increased. The levels of all cytokines remained at control levels until 6 months when the levels of interleukin-6 and -4 were increased. One day after LPS injection, there was a decrease in TH-specific activity that may be due to decreased phosphorylation of serine 31 and 40. This decreased phosphorylation of serine 31 and 40 may be due to an increased activation of the protein phosphatase PP2A. One week after LPS injection, there was increased TH protein and increased phosphorylation of serine 40 that this was not accompanied by an increase in TH-specific activity. All TH parameters measured returned to basal levels between 1 month and 3 months. Six months after injection there was an increase in TH protein. This was associated with increased levels of the extracellular regulated kinase isoforms 1 and 2. This work shows that a single inflammatory event has the capacity to generate both short-term and long-term changes in TH regulation in the adrenal medulla of the adult animal. J. Cell. Biochem. 118: 2096-2107, 2017. © 2016 Wiley Periodicals, Inc.

Protein phosphatase 2A regulation of markers of extracellular matrix remodelling in hepatocellular carcinoma cells: functional consequences for tumour invasion.

A hallmark of tumour invasion is breakdown of the extracellular matrix due to dysregulation of the matrix metalloproteinase (MMP) system. While our understanding of how this is regulated by kinase signalling pathways is well established, its counter-regulation by protein phosphatases (PP) is poorly understood. Therefore, we investigated the effect of PP inhibition on markers of extracellular remodelling and how PP2A activity modulated MMP-9 abundance and function of Hep3B cells. Cells were exposed to okadaic acid (OA), tautomycetin and cyclosporin A, and the expression profile determined using PCR. Effects of OA and a protein inhibitor of PP2A, CIP2A, on MMP-9 abundance, PP2A activity and cell migration were investigated using ELISA, promoter constructs, siRNA knockdown and transwell migration assays. OA increased expression and abundance of MMP-9 and the tissue inhibitor of MMP, TIMP-1, without affecting other MMPs, TIMPs and ADAMs. The effect on MMP-9 was mimicked by CIP2A overexpression and knockdown of the PPP2CA catalytic, but not PPP2R1A scaffolding, subunit. Cyclosporin A and PPP1CA silencing did not alter MMP-9 expression, while tautomycetin transiently increased it. Mutation of AP-1, but not NF-κB, binding sites inhibited OA-mediated MMP-9 transcriptional activity. OA and CIP2A decreased PP2A activity and increased cell migration. OA increased MMP-9 by decreasing PP2A activity and PP2Ac, through AP-1 binding sites on the MMP-9 promoter. The functional consequence of this and CIP2A overexpression was increased cell migration. Hence, PP2A inhibition induced a metastatic phenotype through alterations in MMP-9 in Hep3B cells.

DENV up-regulates the HMG-CoA reductase activity through the impairment of AMPK phosphorylation: A potential antiviral target.

Dengue is the most common mosquito-borne viral disease in humans. Changes of lipid-related metabolites in endoplasmic reticulum of dengue virus (DENV) infected cells have been associated with replicative complexes formation. Previously, we reported that DENV infection inhibits HMGCR phosphorylation generating a cholesterol-enriched cellular environment in order to favor viral replication. In this work, using enzymatic assays, ELISA, and WB we found a significant higher activity of HMGCR in DENV infected cells, associated with the inactivation of AMPK. AMPK activation by metformin declined the HMGCR activity suggesting that AMPK inactivation mediates the enhanced activity of HMGCR. A reduction on AMPK phosphorylation activity was observed in DENV infected cells at 12 and 24 hpi. HMGCR and cholesterol co-localized with viral proteins NS3, NS4A and E, suggesting a role for HMGCR and AMPK activity in the formation of DENV replicative complexes. Furthermore, metformin and lovastatin (HMGCR inhibitor) altered this co-localization as well as replicative complexes formation supporting that active HMGCR is required for replicative complexes formation. In agreement, metformin prompted a significant dose-dependent antiviral effect in DENV infected cells, while compound C (AMPK inhibitor) augmented the viral genome copies and the percentage of infected cells. The PP2A activity, the main modulating phosphatase of HMGCR, was not affected by DENV infection. These data demonstrate that the elevated activity of HMGCR observed in DENV infected cells is mediated through AMPK inhibition and not by increase in PP2A activity. Interestingly, the inhibition of this phosphatase showed an antiviral effect in an HMGCR-independent manner. These results suggest that DENV infection increases HMGCR activity through AMPK inactivation leading to higher cholesterol levels in endoplasmic reticulum necessary for replicative complexes formation. This work provides new information about the mechanisms involved in host lipid metabolism during DENV replicative cycle and identifies new potential antiviral targets for DENV replication.

Liguzinediol Enhances the Inotropic Effect of Rat Hearts via Inhibition of Protein Phosphatase (PP1 and PP2A) Activities.

It has been demonstrated that liguzinediol (2,5-dihydroxymethyl-3,6-dimethylpyrazine, LZDO), a derivative of ligustrazine from Ligusticum wallichii Franch, exerts positive inotropy in isolated rat heart mediated by the sarcoplasmic reticulum Ca-ATPase (SERCA2a). Here, we further explore the underlying mechanism of the positive inotropic effect of LZDO in rat hearts. In vivo and ex vivo rat heart experiments, biochemistry, and Western blot techniques were used to analyze the rat heart contractility, and SERCA2a activity, phospholamban (PLB) phosphorylation, and protein phosphatase (PP1 and PP2A) activities in rat left ventricular myocytes, respectively. LZDO (20 mg/kg) significantly increased the inotropy of rat heart in vivo. In isolated rat heart experiments, LZDO (100 μM) restored the decreased inotropy induced by caffeine (0.5 mM); however, calyculin A (4 nM), an inhibitor of PP1 and PP2A, eliminated the inotropic effect of LZDO (100 μM). Moreover, LZDO (1, 10, and 100 μM) significantly enhanced SERCA2a activity and increased the levels of phosphorylated PLB on both serine-16 (Ser-16) and threonine-17 (Thr-17). In addition, LZDO (100 μM) significantly inhibited the activities of PP1 and PP2A. The positive inotropic effects of LZDO on in vivo and ex vivo rat hearts seem to be mediated through inhibition of PP1/PP2A, which may suppress dephosphorylated PLB and enhance SERCA2a activity. LZDO may prove effective in treating heart failure in clinical settings based on its unique biological mechanism.

(DEAD)-box RNA helicase 3 modulates NF-κB signal pathway by controlling the phosphorylation of PP2A-C subunit.

Asp-Glu-Ala-Asp (DEAD)-box RNA helicase 3 (DDX3), an ATP-dependent RNA helicase, is associated with RNA splicing, mRNA export, transcription, translation, and RNA decay. Recent studies revealed that DDX3 participates in innate immune response during virus infection by interacting with TBK1 and regulating the production of IFN-β. In our studies, we demonstrated that DDX3 regulated NF-κB signal pathway. We found that DDX3 knockdown reduced the phosphorylation of p65 and IKK-β and ultimately attenuated the production of inflammatory cytokines induced by poly(I:C) or TNF-α stimulation. The regulatory effect of DDX3 on NF-κB signal pathway was not affected by the loss of its ATPase or helicase activity. We further identified PP2A C subunit (PP2A-C) as an interaction partner of DDX3 by co-immunoprecipitation and mass spectrum analysis. We confirmed that DDX3 formed the complex with PP2A-C/IKK-β and regulated the interaction between IKK-β and PP2A-C. Furthermore, we demonstrated that DDX3 modulated the activity of PP2A by controlling the phosphorylation of PP2A-C, which might enable PP2A-C to regulate NF-κB signal pathway by dephosphorylating IKK-β. All these findings suggested DDX3 plays multiple roles in modulating innate immune system.

Systematic Analysis of Human Protein Phosphatase Interactions and Dynamics

Coordinated activities of protein kinases and phosphatases ensure phosphorylation homeostasis, which, when perturbed, can instigate diseases, including cancer. Yet, in contrast to kinases, much less is known about protein phosphatase functions and their interactions and complexes. Here, we used quantitative affinity proteomics to assay protein-protein interactions for 54 phosphatases distributed across the three major protein phosphatase families, with additional analysis of their 12 co-factors. We identified 838 high-confidence interactions, of which 631, to our knowledge, have not been reported before. We show that inhibiting the activity of phosphatases PP1 and PP2A by okadaic acid disrupts their specific interactions, supporting the potential of therapeutics that target these proteins. Additional analyses revealed candidate physical and functional interaction links to phosphatase-based regulation of several signaling pathways and to human cancer. Our study provides an initial glimpse of the protein interaction landscape of phosphatases and their functions in cellular regulation.

Ex vivo modulation of the Foxo1 phosphorylation state does not lead to dysfunction of T regulatory cells.

Peripheral regulatory CD4+ T cells (Treg cells) prevent maladaptive inflammatory responses to innocuous foreign antigens. Treg cell dysfunction has been linked to many inflammatory diseases, including allergic airway inflammation. Glucocorticoids that are used to treat allergic airway inflammation and asthma are thought to work in part by promoting Treg cell differentiation; patients who are refractory to these drugs have defective induction of anti-inflammatory Treg cells. Previous observations suggest that Treg cells deficient in the transcription factor FoxO1 are pro-inflammatory, and that FoxO1 activity is regulated by its phosphorylation status and nuclear localization. Here, we asked whether altering the phosphorylation state of FoxO1 through modulation of a regulatory phosphatase might affect Treg cell function. In a mouse model of house dust mite-induced allergic airway inflammation, we observed robust recruitment of Treg cells to the lungs and lymph nodes of diseased mice, without an apparent increase in the Treg cytokine interleukin-10 in the airways. Intriguingly, expression of PP2A, a serine/threonine phosphatase linked to the regulation of FoxO1 phosphorylation, was decreased in the mediastinal lymph nodes of HDM-treated mice, mirroring the decreased PP2A expression seen in peripheral blood monocytes of glucocorticoid-resistant asthmatic patients. When we asked whether modulation of PP2A activity alters Treg cell function via treatment with the PP2A inhibitor okadaic acid, we observed increased phosphorylation of FoxO1 and decreased nuclear localization. However, dysregulation of FoxO1 did not impair Treg cell differentiation ex vivo or cause Treg cells to adopt a pro-inflammatory phenotype. Moreover, inhibition of PP2A activity did not affect the suppressive function of Treg cells ex vivo. Collectively, these data suggest that modulation of the phosphorylation state of FoxO1 via PP2A inhibition does not modify Treg cell function ex vivo. Our data also highlight the caveat in using ex vivo assays of Treg cell differentiation and function, in that while these assays are useful, they may not fully recapitulate Treg cell phenotypes that are observed in vivo.

The protective effects of propofol against CoCl2-induced HT22 cell hypoxia injury via PP2A/CAMKII\u03b1/nNOS pathway

BackgroundPerioperative cerebral ischemia/hypoxia could induce hippocampal injury and has been reported to induce cognitive impairment. In this study, we used cobalt chloride (CoCl2) to build a hypoxia model in mouse hippocampal cell lines. Propofol, a widely used intravenous anesthetic agent, has been demonstrated to have neuroprotective effect. Here, we explored whether and how propofol attenuated CoCl2-induced mouse hippocampal HT22 cell injury.MethodsMouse hippocampal HT22 cells were pretreated with propofol, and then stimulated with CoCl2. Cell viability was measured by cell counting kit 8 (CCK8). The effect of propofol on CoCl2-modulated expressions of B-cell lymphoma 2 (Bcl-2), BAX, cleaved caspase 3, phosphatase A2 (PP2A), and the phosphorylation of Ca2+/Calmodulin (CaM)-dependent protein kinase II (pCAMKIIα), neuron nitric oxide synthase at Ser1412 (pnNOS-Ser1412), neuron nitric oxide synthase at Ser847 (pnNOS-Ser847) were detected by Western blot analysis.ResultsCompared with control, CoCl2 treatment could significantly decrease cell viability, which could be reversed by propofol. Further, we found CoCl2 treatment could up-regulate the expression of PP2A, BAX, cleaved caspase three and cause the phosphorylation of nNOS-Ser1412, but it down-regulated the expression of Bcl-2 and the phosphorylation of CAMKIIα and nNOS-Ser847. More importantly, these CoCl2-mediated effects were attentuated by propofol. In addition, we demonstrated that propofol could exert similar effect to that of the PP2A inhibitor (okadaic acid). Further, the PP2A activator (FTY720) and the CAMKIIα inhibitor (KN93) could reverse the neuroprotective effect of propofol.ConclusionOur data indicated that propofol could attenuate CoCl2-induced HT22 cells hypoxia injury via PP2A/CAMKIIα/nNOS pathway.

Cancer-associated Fibroblasts Promote Irradiated Cancer Cell Recovery Through Autophagy

Tumor relapse after radiotherapy is a significant challenge to oncologists, even after recent the advances in technologies. Here, we showed that cancer-associated fibroblasts (CAFs), a major component of cancer stromal cells, promoted irradiated cancer cell recovery and tumor relapse after radiotherapy. We provided evidence that CAFs-produced IGF1/2, CXCL12 and β-hydroxybutyrate were capable of inducing autophagy in cancer cells post-radiation and promoting cancer cell recovery from radiation-induced damage in vitro and in vivo in mice. These CAF-derived molecules increased the level of reactive oxygen species (ROS) post-radiation, which enhanced PP2A activity, repressing mTOR activation and increasing autophagy in cancer cells. Consistently, the IGF2 neutralizing antibody and the autophagy inhibitor 3-MA reduce the CAF-promoted tumor relapse in mice after radiotherapy. Taken together, our findings demonstrated that CAFs promoted irradiated cancer cell recovery and tumor regrowth post-radiation, suggesting that targeting the autophagy pathway in tumor cells may be a promising therapeutic strategy for radiotherapy sensitization.

Propofol inhibits high glucose-induced PP2A expression in human umbilical vein endothelial cells

Perioperative hyperglycemia is a common clinical metabolic disorder. Hyperglycemia could induce endothelial apoptosis, dysfunction and inflammation, resulting in endothelial injury. Propofol is a widely used anesthetic drug in clinical settings. Our previous studies indicated that propofol, via inhibiting high glucose-induced phosphatase A2 (PP2A) expression, attenuated high glucose-induced reactive oxygen species (ROS) accumulation, thus improving endothelial apoptosis, dysfunction and inflammation. However, the mechanisms by which propofol attenuated high glucose-induced PP2A expression is still obscure. In the present study, we examined how propofol attenuates high glucose-induced endothelial PP2A expression. Compared with 5mM glucose treatment, 15mM glucose up-regulated expression and activity of PP2A, increased cAMP response element binding protein (CREB), Ca2+-calmodulin dependent kinase II (CaMK II) phosphorylation and Ca2+ accumulation. More importantly, propofol decreased PP2A expression and activity, attenuated CREB, CaMK II phosphorylation and Ca2+ accumulation in a concentration-dependent manner. Moreover, we demonstrated that the effect of propofol was similar to that of MK801, an inhibitor of NMDA receptor. In contrast, rapastinel, an activator of NMDA receptor, antagonized the effect of propofol. Also, the effect of KN93, an inhibitor of CaMK II, was similar to that of propofol, except KN93 had no effect on 15mM glucose-mediated Ca2+ accumulation. Our data indicated that propofol, via inhibiting NMDA receptor, attenuated 15mM glucose-induced Ca2+ accumulation, CaMK II and CREB phosphorylation, thus inhibiting PP2A expression and improving 15mM glucose-induced endothelial dysfunction and inflammation.

Active β-catenin is regulated by the PTEN/PI3 kinase pathway: a role for protein phosphatase PP2A.

Dysregulation of Wnt/β-catenin signaling has been associated with the development and progression of many cancers. The stability and subcellular localization of β-catenin, a dual functional protein that plays a role in intracellular adhesion and in regulating gene expression, is tightly regulated. However, little is known about the transcriptionally active form of β-catenin, Active Beta Catenin (ABC), that is unphosphorylated at serine 37 (Ser37) and threonine 41 (Thr41). Elucidating the mechanism by which β-catenin is activated to generate ABC is vital to the development of therapeutic strategies to block β-catenin signaling for cancer treatment. Using melanoma, breast and prostate cancer cell lines, we show that while cellular β-catenin levels are regulated by the Wnt pathway, cellular ABC levels are mainly regulated by the PI3K pathway and are dependent on the phosphatase activity of the protein phosphatase PP2A. Furthermore, we demonstrate that although the PI3K/PTEN pathway does not regulate total β-catenin protein levels within the cell, it plays a role in regulating the subcellular localization of β-catenin. Our results support a novel functional interaction/cross-talk between the PTEN/PI3K and Wnt pathways in the regulation of the subcellular/nuclear levels of ABC, which is crucially important for the protein's activity as a transcription factor and its biological effects in health and disease.