Optogenetically engineered calcium oscillations promote autophagy-mediated cell death via AMPK activation.

Autophagy is a double-edged sword for cells; it can lead to both cell survival and death. Calcium (Ca2+) signalling plays a crucial role in regulating various cellular behaviours, including cell migration, proliferation and death. In this study, we investigated the effects of modulating cytosolic Ca2+ levels on autophagy using chemical and optogenetic methods. Our findings revealed that ionomycin and thapsigargin induce Ca2+ influx to promote autophagy, whereas the Ca2+ chelator BAPTA-AM induces Ca2+ depletion and inhibits autophagy. Furthermore, the optogenetic platform allows the manipulation of illumination parameters, including density, frequency, duty cycle and duration, to create different patterns of Ca2+ oscillations. We used the optogenetic tool Ca2+-translocating channelrhodopsin, which is activated and opened by 470 nm blue light to induce Ca2+ influx. These results demonstrated that high-frequency Ca2+ oscillations induce autophagy. In addition, autophagy induction may involve Ca2+-activated adenosine monophosphate (AMP)-activated protein kinases. In conclusion, high-frequency optogenetic Ca2+ oscillations led to cell death mediated by AMP-activated protein kinase-induced autophagy.

In vitro effect of diazoxon on cell signaling and second messengers in Nile tilapia (Oreochromis niloticus) leukocytes.

The physiological and molecular responses of leukocytes are altered by organophosphate pesticides. Some reports have shown that diazinon causes immunotoxic effects; diazoxon, the oxon metabolite of diazinon, is attributed to influence the immune response by affecting the leukocyte cholinergic system. In this study, the in vitro effects of diazoxon on molecules involved in cell signaling (cAMP, IP3, DAG, JAK1, and STAT3), which play a crucial role in the activation, differentiation, and survival of leukocytes, were evaluated. Data indicate that diazoxon leads to a decrease in cAMP concentration and an increase in basal IP3 levels. However, diazoxon does not affect basal levels of JAK1 and STAT3 phosphorylation. Instead, diazoxon inhibits leukocyte responsiveness to phorbol myristate acetate and ionomycin, substances that, under normal conditions, enhance JAK/STAT signaling. These findings demonstrate that diazoxon significantly affects key molecular parameters related to cell signaling.

Macrophage extracellular traps require peptidylarginine deiminase 2 and 4 and are a source of citrullinated antigens bound by rheumatoid arthritis autoantibodies.

Introduction: Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis, but the sources of citrullinated antigens as well as which peptidylarginine deiminases (PADs) are required for their production remain incompletely defined. Here, we investigated if macrophage extracellular traps (METs) could be a source of citrullinated proteins bound by APCAs, and if their formation requires PAD2 or PAD4. Methods: Thioglycolate-induced peritoneal macrophages from wild-type, PAD2-/-, and PAD4-/- mice or human peripheral blood-derived M1 macrophages were activated with a variety of stimulants, then fixed and stained with DAPI and either anti-citrullinated histone H4 (citH4) antibody or sera from ACPA+ or ACPA- rheumatoid arthritis subjects. METs were visualized by immunofluorescence, confirmed to be extracellular using DNase, and quantified. Results: We found that ionomycin and monosodium urate crystals reliably induced murine citH4+ METs, which were reduced in the absence of PAD2 and lost in the absence of PAD4. Also, IgG from ACPA+, but not ACPA-, rheumatoid arthritis sera bound to murine METs, and in the absence of PAD2 or PAD4, ACPA-bound METs were lost. Finally, ionomycin induced human METs that are citH4+ and ACPA-bound. Discussion: Thus, METs may contribute to the pool of citrullinated antigens bound by ACPAs in a PAD2- and PAD4-dependent manner, providing new insights into the targets of immune tolerance loss in rheumatoid arthritis.

Adoptive immunotherapy with cells from tumor-draining lymph nodes activated and expanded in vitro.

Tumor-draining lymph nodes (tumor-DLNs) provide a rich source of tumor-reactive lymphocytes which can be used in adoptive immunotherapy (AIT) and that circumvent the need to resect autologous tumor, without the challenges and shortcomings associated with using autologous tumor or anti-CD3 monoclonal antibody. Bryostatin/Ionomycin (Bryo/Io) provide a useful method of activating tumor-DLNs such that they can readily be expanded to sufficient numbers to be used in AIT, and growing the tumor-DLN lymphocytes in the gamma chain cytokines IL-7 plus IL-15 is superior to IL-2 in terms of T cell numbers and phenotype. AIT with these cells induces tumor regression and provides protection against metastases and future tumor challenge. Here, we provide a stepwise protocol to sensitize tumor-DLN cells in donor mice, activate tumor-DLN T cells ex vivo using Bryo/Io, expansion of these cells in gamma chain cytokines and adoptive transfer of the expanded cells back into tumor-bearing hosts. Methods relevant to these experiments, such as injecting tumor cells intravenously and monitoring for pulmonary metastases, tumor volume measurement and resection, and use of luciferase-expressing tumor cells to monitor for metastases following resection, are described in detail. The methods outlined herein can be easily adapted to suit similar experiments across multiple tumor cell lines and syngeneic mouse models.

Canine T zone lymphoma is a tumor of mature, previously activated αß T cells.

T cell lymphomas are a diverse group of tumors found in both dogs and humans, originating from various normal T cell types. Identifying the origin of neoplastic lymphocytes can offer valuable insights into the pathogenesis and clinical behavior of these tumors. T zone lymphoma (TZL) in dogs is characterized by the absence of CD45 expression, a strong breed predilection, and its association with adult-onset demodicosis-a condition believed to be linked to immunosuppression. In this study, our aim was to employ transcriptomic and functional data to determine the normal counterpart of TZL. Identifying the normal counterpart may help us understand both how these tumors arise and explain their clinical behavior. Gene expression profiling using NanoString and RNA seq was used to compare the transcriptome between neoplastic T zone cells, normal canine T cells and publicly available gene sets using Gene Set Enrichment Analysis. Mitogen, anti-CD3 stimulation and PMA/ionomycin stimulation were used to assess T cell proliferation in vitro, and intracellular cytokine production was measured by flow cytometry. Gene expression profiling revealed that TZL is most likely derived from an activated or memory alpha-beta T cell but the cells do not fall cleanly into an effector subtype. TZL cells express CD4-specific transcription factors GATA3 and THPOK, even though TZL cells more commonly express CD8, or neither CD4 nor CD8. TZL cells produce high levels of interferon gamma and tumor necrosis factor alpha when stimulated, further supporting the hypothesis that they are derived from an antigen experienced T cell. TZL cells do not proliferate when stimulated through the T cell receptor but will divide when the T cell receptor is bypassed with PMA and ionomycin. The observation that these cells are derived from a mature, previously activated T cell is the first step in understanding the genesis of this unique T cell tumor.

Characterization of two distinct immortalized endothelial cell lines, EA.hy926 and HMEC-1, for in vitro studies: exploring the impact of calcium electroporation, Ca2+ signaling and transcriptomic profiles.

BACKGROUND: Disruption of Ca2+ homeostasis after calcium electroporation (CaEP) in tumors has been shown to elicit an enhanced antitumor effect with varying impacts on healthy tissue, such as endothelium. Therefore, our study aimed to determine differences in Ca2+ kinetics and gene expression involved in the regulation of Ca2+ signaling and homeostasis, as well as effects of CaEP on cytoskeleton and adherens junctions of the established endothelial cell lines EA.hy926 and HMEC-1. METHODS: CaEP was performed on EA.hy926 and HMEC-1 cells with increasing Ca2+ concentrations. Viability after CaEP was assessed using Presto Blue, while the effect on cytoskeleton and adherens junctions was evaluated via immunofluorescence staining (F-actin, α-tubulin, VE-cadherin). Differences in intracellular Ca2+ regulation ([Ca2+]i) were determined with spectrofluorometric measurements using Fura-2-AM, exposing cells to DPBS, ionomycin, thapsigargin, ATP, bradykinin, angiotensin II, acetylcholine, LaCl3, and GdCl3. Molecular distinctions were identified by analyzing differentially expressed genes and pathways related to the cytoskeleton and Ca2+ signaling through RNA sequencing. RESULTS: EA.hy926 cells, at increasing Ca2+ concentrations, displayed higher CaEP susceptibility and lower survival than HMEC-1. Immunofluorescence confirmed CaEP-induced, time- and Ca2+-dependent morphological changes in EA.hy926's actin filaments, microtubules, and cell-cell junctions. Spectrofluorometric Ca2+ kinetics showed higher amplitudes in Ca2+ responses in EA.hy926 exposed to buffer, G protein coupled receptor agonists, bradykinin, and angiotensin II compared to HMEC-1. HMEC-1 exhibited significantly higher [Ca2+]i changes after ionomycin exposure, while responses to thapsigargin, ATP, and acetylcholine were similar in both cell lines. ATP without extracellular Ca2+ ions induced a significantly higher [Ca2+]i rise in EA.hy926, suggesting purinergic ionotropic P2X and metabotropic P2Y receptor activation. RNA-sequencing analysis showed significant differences in cytoskeleton- and Ca2+-related gene expression, highlighting upregulation of ORAI2, TRPC1, TRPM2, CNGA3, TRPM6, and downregulation of TRPV4 and TRPC4 in EA.hy926 versus HMEC-1. Moreover, KEGG analysis showed upregulated Ca2+ import and downregulated export genes in EA.hy926. CONCLUSIONS: Our finding show that significant differences in CaEP response and [Ca2+]i regulation exist between EA.hy926 and HMEC-1, which may be attributed to distinct transcriptomic profiles. EA.hy926, compared to HMEC-1, displayed higher susceptibility and sensitivity to [Ca2+]i changes, which may be linked to overexpression of Ca2+-related genes and an inability to mitigate changes in [Ca2+]i. The study offers a bioinformatic basis for selecting EC models based on research objectives.

Methiothepin downregulates SNAP-23 and inhibits degranulation of rat basophilic leukemia cells and mouse bone marrow-derived mast cells.

In the present study, we found that methiothepin (a nonselective 5-hydroxytryptamine [5-HT] receptor antagonist) inhibited antigen-induced degranulation in rat basophilic leukemia cells and mouse bone marrow-derived mast cells. Although antigen stimulation induces release of histamine and serotonin (5-HT) by exocytosis and mast cells express several types of 5-HT receptor, the detailed role of these receptors remains unclear. Here, pretreatment of cells with methiothepin attenuated increased intracellular Ca2+ concentration, phosphorylated critical upstream signaling components (Src family tyrosine kinases, Syk, and PLCγ1), and suppressed TNF-α secretion via inhibition of Akt (a Ser/Thr kinase activated by PI3K)and ERK phosphorylation. Furthermore, it inhibited PMA/ionomycin-induced degranulation; this finding suggested that methiothepin affected downstream signaling. IκB kinase ß phosphorylates synaptosomal associated protein 23, which regulates the fusion events of the secretory granule/plasma membrane after mast cell activation, resulting in degranulation. We showed that methiothepin blocked PMA/ionomycin-induced phosphorylation of synaptosomal associated protein 23 by inhibiting its interaction with IκB kinase ß. Together with the results of selective 5-HT antagonists, it is suggested that methiothepin inhibits mast cell degranulation by downregulating upstream signaling pathways and exocytotic fusion machinery through mainly 5-HT1A receptor. Our findings provide that 5-HT antagonists may be used to relieve allergic reactions.

Mechanical Properties Affect Primary T Cell Activation in 3D Bioprinted Hydrogels.

T cells play a critical role in the adaptive immune response of the body, especially against intracellular pathogens and cancer. In vitro, T cell activation studies typically employ planar (two-dimensional, 2D) culture systems that do not mimic native cell-to-extracellular matrix (ECM) interactions, which influence activation. The goal of this work was to study T cell responses in a cell line (EL4) and primary mouse T cells in three-dimensional (3D) bioprinted matrices of varied stiffness. Cell-laden hydrogels were 3D bioprinted from gelatin methacryloyl (GelMA) using a digital light processing (DLP)-based 3D bioprinter operated with visible light (405 nm). Mechanical characterization revealed that the hydrogels had pathophysiologically relevant stiffnesses for a lymph node-mimetic tissue construct. EL4, a mouse T cell lymphoma line, or primary mouse T cells were 3D bioprinted and activated using a combination of 10 ng/mL of phorbol myristate acetate (PMA) and 0.1 µM of ionomycin. Cellular responses revealed differences between 2D and 3D cultures and that the biomechanical properties of the 3D bioprinted hydrogel influence T cell activation. Cellular responses of the 2D and 3D cultures in a soft matrix (19.83 ± 2.36 kPa) were comparable; however, they differed in a stiff matrix (52.95 ± 1.36 kPa). The fraction of viable EL4 cells was 1.3-fold higher in the soft matrix than in the stiff matrix. Furthermore, primary mouse T cells activated with PMA and ionomycin showed 1.35-fold higher viable cells in the soft matrix than in the stiff matrix. T cells bioprinted in a soft matrix and a stiff matrix released 7.4-fold and 5.9-fold higher amounts of interleukin-2 (IL-2) than 2D cultured cells, respectively. Overall, the study demonstrates the changes in the response of T cells in 3D bioprinted scaffolds toward engineering an ex vivo lymphoid tissue-mimetic system that can faithfully recapitulate T cell activation and unravel pathophysiological characteristics of T cells in infectious biology, autoimmunity, and cancers.

An increase in the cytosolic concentration of free calcium ions activates the neutrophil NADPH-oxidase provided that the free fatty acid receptor 2 has been allosterically modulated.

Signals generated by free fatty acid receptor 2 (FFA2R) can activate the neutrophil NADPH-oxidase without involvement of any orthosteric FFA2R agonist. The initiating signals may be generated by P2Y2R, the receptor for ATP. An FFA2R specific allosteric modulator (PAM; Cmp58) was required for this response and used to investigate the mechanism by which signals generated by ATP/P2Y2R activate an FFA2R dependent process. The P2Y2R induced signal that together with the modulated FFA2R activates neutrophils, was generated downstream of the Gαq containing G protein coupled to P2Y2R. A rise in the cytosolic concentration of ionized calcium ([Ca2+]i) was hypothesized to be the important signal. The hypothesis gained support from the finding that the modulator transferred the neutrophils to a Ca2+sensitive state. The rise in [Ca2+]i induced by the Ca2+ specific ionophore ionomycin, activated the neutrophils provided that an allosteric modulator was bound to FFA2R. The activity of the superoxide generating NADPH-oxidase induced by ionomycin was rapidly terminated and the FFA2Rs could then no longer be activated by the FFA2R agonist propionate or by the signal generated by ATP/P2Y2R. The non-responding state of FFA2R was, however, revoked by a cross-activating allosteric FFA2R modulator. The [Ca2+]i mediated activation of neutrophils with their FFA2Rs allosterically modulated, represent a unique regulatory receptor crosstalk mechanism by which the activation potency of a G protein coupled receptor is controlled by a receptor-crosstalk signaling system operating from the cytosolic side of the plasma membrane.

Dysregulation of ADAM10 shedding activity in naked mole-rat fibroblasts is due to deficient phosphatidylserine externalization.

The naked mole-rat (NMR, Heterocephalus glaber) is of significant interest to biogerontological research, rarely developing age-associated diseases, such as cancer. The transmembrane glycoprotein CD44 is upregulated in certain cancers and CD44 cleavage by a disintegrin and metalloproteinase 10 (ADAM10) regulates cellular migration. Here we provide evidence that mature ADAM10 is expressed in NMR primary skin fibroblasts (NPSF), and that ionomycin increases cell surface ADAM10 localization. However, we observed an absence of ADAM10 mediated CD44 cleavage, as well as shedding of exogenous and overexpressed betacellulin in NPSF, whereas in mouse primary skin fibroblasts ionomycin induced ADAM10-dependent cleavage of both CD44 and betacellulin. Overexpressing a hyperactive form of the Ca2+ -dependent phospholipid scramblase ANO6 in NPSF increased phosphatidylserine (PS) externalization, which rescued the ADAM10 sheddase activity and promoted cell migration in NPSF in an ADAM10-dependent manner. These findings suggest that dysregulation of ADAM10 shedding activity is due to a deficient PS externalization in NMR.

Usage of celery root exosome as an immune suppressant; Lipidomic characterization of apium graveolens originated exosomes and its suppressive effect on PMA/ionomycin mediated CD4+ T lymphocyte activation.

Diseases such as autoimmune, cancer, neurodegenerative diseases or obesity have a serious impact on the lives of patients all rise from a common point; the immune system. Various in vitro and in vivo studies on regulating the immune system have been made to correct these diseases. As one of the key effector cells of the immune system, T lymphocytes are the focus of many of these studies. In this study, exosomes isolated from a known anti-inflammatory plant, celery, were used to suppress the inflammatory response of T lymphocytes. Celery-derived exosomes (C-Exo) were isolated using an aqueous two-phase isolation method. The size distribution, morphology, particle concentration, and GC-FAME-based lipidomic analysis were determined for the isolated C-Exo. T lymphocytes were stimulated using Phorbol 12-myristate 13-acetate (PMA)/ionomycin, and treated with various doses of C-Exo. T lymphocyte responses were measured using qPCR and capillary Western blots. According to the results, C-Exo suppressed T lymphocytes in a dose-dependent manner in in vitro conditions. These findings show the potential of C-Exo as a therapeutic agent for immune disorders. PRACTICAL APPLICATION: Excessive immune response in the body adversely affects the treatment mechanism and process of many diseases such as autoimmune disorders, neurodegenerative diseases and GDHV. In this preliminary study, the role of extracellular vesicles obtained from celery roots in suppressing this high immune response was investigated. The suppressive effect of celery exosome was observed by creating an immune response in T cells and PBMC cells, which play a leading role in the immune response. The role of these vesicles in immune suppression, obtained from the root part of the celery plant and characterized, was determined by measuring both mRNA, intracellular protein and extracellular cytokine levels. Celery exosome suppressed activated T lymphocyte cells and PBMC cells in a dose-dependent manner. These vesicles, which can be used as an edible, can be used in many areas as immunosuppressants.

Inhibition of p38 MAPK or immunoproteasome overcomes resistance of chronic lymphocytic leukemia cells to Bcl-2 antagonist venetoclax.

Chronic lymphocytic leukemia (CLL) is a hematological neoplasm of CD19-positive mature-appearing B lymphocytes. Despite the clinical success of targeted therapies in CLL, the development of resistance diminishes their therapeutic activity. This is also true for the Bcl-2 antagonist venetoclax. We investigated the molecular mechanisms that drive venetoclax resistance in CLL, with a clear focus to provide new strategies to successfully combat it. Activation of CLL cells with IFNγ, PMA/ionomycin, and sCD40L diminished the cytotoxicity of venetoclax. We demonstrated that the metabolic activity of cells treated with 1 nM venetoclax alone was 48% of untreated cells, and was higher for cells co-treated with IFNγ (110%), PMA/ionomycin (78%), and sCD40L (62%). As of molecular mechanism, we showed that PMA/ionomycin and sCD40L triggered translocation of NFκB in primary CLL cells, while IFNγ activated p38 MAPK, suppressed spontaneous and venetoclax-induced apoptosis and induced formation of the immunoproteasome. Inhibition of immunoproteasome with ONX-0914 suppressed activity of immunoproteasome and synergized with venetoclax against primary CLL cells. On the other hand, inhibition of p38 MAPK abolished cytoprotective effects of IFNγ. We demonstrated that venetoclax-resistant (MEC-1 VER) cells overexpressed p38 MAPK and p-Bcl-2 (Ser70), and underexpressed Mcl-1, Bax, and Bak. Inhibition of p38 MAPK or immunoproteasome triggered apoptosis in CLL cells and overcame the resistance to venetoclax of MEC-1 VER cells and venetoclax-insensitive primary CLL cells. In conclusion, the p38 MAPK pathway and immunoproteasome represent novel targets to combat venetoclax resistance in CLL.

Human Amniotic Membrane Mesenchymal Stem Cell-Synthesized PGE2 Exerts an Immunomodulatory Effect on Neutrophil Extracellular Trap in a PAD-4-Dependent Pathway through EP2 and EP4.

Human amniotic membrane mesenchymal stem cells (hAM-MSC) secrete a myriad of components with immunosuppressive activities. In the present research, we aimed to describe the effect of prostaglandin E2 (PGE2) secreted by hAM-MSCs on neutrophil extracellular trap (NET) release and to characterize the role of its receptors (EP2/EP4) in PAD-4 and NFκB activity in neutrophils. Human peripheral blood neutrophils were ionomycin-stimulated in the presence of hAM-MSC conditioned medium (CM) treated or not with the selective PGE2 inhibitor MF-63, PGE2, EP2/EP4 agonists, and the selective PAD-4 inhibitor GSK-484. NET release, PAD-4, and NFκB activation were analyzed. Ionomycin induced NET release, which was inhibited in the presence of hAM-MSC-CM, while CM from hAM-MSCs treated with MF-63 prevented NET release inhibition. PGE2 and EP2/EP4 agonists, and GSK-484 inhibited NET release. EP2/EP4 agonists and GSK-484 inhibited H3-citrullination but did not affect PAD-4 protein expression. Finally, PGE2 and EP2/EP4 agonists and GSK-484 increased NFκB phosphorylation. Taken together, these results suggest that hAM-MSC exert their immunomodulatory activities through PGE2, inhibiting NET release in a PAD-4-dependent pathway. This research proposes a new mechanism by which hAM-MSC exert their activities when modulating the innate immune response and inhibiting NET release.

Design, synthesis and in vitro biological evaluation of marine phidianidine derivatives as potential anti-inflammatory agents.

Phidianidines A and B are novel marine indole alkaloids with various biological activities. Based on their potential anti-inflammatory properties, a series of phidianidine derivatives were designed, synthesized, and tested for their effects on IL-17A production in PMA/ionomycin-stimulated T-cell-lymphoma EL-4 cells. Compounds 9a and 22c exhibited excellent anti-inflammatory activity and low toxicity, with IC50 values of 7.7 µM and 5.3 µM for IL-17A production in PMA/ionomycin-stimulated EL-4 cells, respectively. Further mechanistic study showed that 9a could decrease the STAT3 phosphorylation at Y705 to inhibit IL-17A production in EL-4 cells, indicating its ability of preventing the differentiation of Th17 cells and their possible function. This research may give an insight for the discovery of marine indole alkaloid derived anti-inflammatory drug leads for the treatment of T cell-mediated diseases.

Phosphatidylinositol Monophosphates Regulate the Membrane Localization of HSPA1A, a Stress-Inducible 70-kDa Heat Shock Protein.

HSPA1A is a molecular chaperone that regulates the survival of stressed and cancer cells. In addition to its cytosolic pro-survival functions, HSPA1A also localizes and embeds in the plasma membrane (PM) of stressed and tumor cells. Membrane-associated HSPA1A exerts immunomodulatory functions and renders tumors resistant to standard therapies. Therefore, understanding and manipulating HSPA1A's surface presentation is a promising therapeutic. However, HSPA1A's pathway to the cell surface remains enigmatic because this protein lacks known membrane localization signals. Considering that HSPA1A binds to lipids, like phosphatidylserine (PS) and monophosphorylated phosphoinositides (PIPs), we hypothesized that this interaction regulates HSPA1A's PM localization and anchorage. To test this hypothesis, we subjected human cell lines to heat shock, depleted specific lipid targets, and quantified HSPA1A's PM localization using confocal microscopy and cell surface biotinylation. These experiments revealed that co-transfection of HSPA1A with lipid-biosensors masking PI(4)P and PI(3)P significantly reduced HSPA1A's heat-induced surface presentation. Next, we manipulated the cellular lipid content using ionomycin, phenyl arsine oxide (PAO), GSK-A1, and wortmannin. These experiments revealed that HSPA1A's PM localization was unaffected by ionomycin but was significantly reduced by PAO, GSK-A1, and wortmannin, corroborating the findings obtained by the co-transfection experiments. We verified these results by selectively depleting PI(4)P and PI(4,5)P2 using a rapamycin-induced phosphatase system. Our findings strongly support the notion that HSPA1A's surface presentation is a multifaceted lipid-driven phenomenon controlled by the binding of the chaperone to specific endosomal and PM lipids.

Substrate recognition by Arg/Pro-rich insert domain in calcium/calmodulin-dependent protein kinase kinase for target protein kinases.

Calcium/calmodulin-dependent protein kinase kinases (CaMKKs) activate CaMKI, CaMKIV, protein kinase B/Akt, and AMP-activated protein kinase (AMPK) by phosphorylating Thr residues in activation loops to mediate various Ca2+ -signaling pathways. Mammalian cells expressing CaMKKα and CaMKKß lacking Arg/Pro-rich insert domain (RP-domain) sequences showed impaired phosphorylation of AMPKα, CaMKIα, and CaMKIV, whereas the autophosphorylation activities of CaMKK mutants remained intact and were similar to those of wild-type CaMKKs. Liver kinase B1 (LKB1, an AMPK kinase) complexed with STRAD and MO25 and was unable to phosphorylate CaMKIα and CaMKIV; however, mutant LKB1 with the RP-domain sequences of CaMKKα and CaMKKß inserted between kinase subdomains II and III acquired CaMKIα and CaMKIV phosphorylating activity in vitro and in transfected cultured cells. Furthermore, ionomycin-induced phosphorylation of hemagglutinin (HA)-CaMKIα at Thr177, HA-CaMKIV at Thr196, and HA-AMPKα at Thr172 in transfected cells was significantly suppressed by cotransfection of kinase-dead mutants of CaMKK isoforms, but these dominant-negative effects were abrogated with RP-deletion mutants, suggesting that sequestration of substrate kinases by loss-of-function CaMKK mutants requires the RP-domain. This was confirmed by pulldown experiments that showed that dominant-negative mutants of CaMKKα and CaMKKß interact with target kinases but not RP-deletion mutants. Taken together, these results clearly indicate that both CaMKK isoforms require the RP-domain to recognize downstream kinases to interact with and phosphorylate Thr residues in their activation loops. Thus, the RP-domain may be a promising target for specific CaMKK inhibitors.

Protein phosphatase 2A catalytic subunit ß suppresses PMA/ionomycin-induced T-cell activation by negatively regulating PI3K/Akt signaling.

The precise regulation of the T-cell activation process is critical for overall immune homeostasis. Although protein phosphatase 2A (PP2A) is required for T-cell development and function, the role of PPP2CB, which is the catalytic subunit ß isoform of PP2A, remains unknown. In the present study, using a T cell-specific knockout mouse of PPP2CB (PPP2CBfl/fl Lck-Cre+ ), we demonstrated that PPP2CB was dispensable for T-cell development in the thymus and peripheral lymphoid organs. Furthermore, PPP2CB deletion did not affect T-cell receptor (TCR)-induced T-cell activation or cytokine-induced T-cell responses; however, it specifically enhanced phorbol myristate acetate (PMA) plus ionomycin-induced T-cell activation with increased cellular proliferation, elevated CD69 and CD25 expression, and enhanced cytokine production (inteferon-γ, interleukin-2 and tumor necrosis factor). Mechanistic analyses suggested that the PPP2CB deletion enhanced activation of the phosphoinositide 3-kinase/Akt signaling pathway and Ca2+ flux following stimulation with PMA plus ionomycin. Moreover, the specific PI3K inhibitor rescued the augmented cell activation in PPP2CB-deficient T cells. Using mass spectrometry-based phospho-peptide analysis, we identified potential substrates of PPP2CB during PMA plus ionomycin-induced T-cell activation. Collectively, our study provides evidence of the specific role of PPP2CB in controlling PMA plus ionomycin-induced T-cell activation.

Inhibition of Histamine Release from RBL-2H3 Cells by Zoledronate Did Not Affect Rab27a/Doc2a Interaction.

Mast cell (MC) exocytosis is organized by prenylated protein, including Rab families. Among Rab proteins, Rab3a, Rab27a, and Rab11 are responsible for exocytosis arrangement. Rab3a and Rab27a are contributed to exocytosis by interacting with other exocytosis proteins. Zoledronate administration disrupted the Rab prenylation process that affected its interaction with other proteins, and finally, its function. The present study has investigated the effect of zoledronate on the histamine release (HR) from RBL-2H3 cells. The main focus is to answer the question of whether zoledronate affects Rab27a/Doc2a interaction. Histamine release on RBL-2H3 cells after zoledronate or clodronate administration was measured using HPLC-fluorometry. Dinitrophenylated bovine serum albumin (DNP-BSA) (20 ng/mL) or ionomycin (1 µM) are used as secretagogues. Calcium (Ca2+) influx observation was performed using Fura-2A/M. In situ proximity ligation assay (PLA) is used to investigate Rab27a/Doc2a interaction after bisphosphonates (BPs) treatment. Histamine concentration measurement with HPLC-fluorometry showed that zoledronate (30, 100 µM) inhibited HR from antigen-activated RBL-2H3 cells. Zoledronate showed less inhibition in cells activated with ionomycin. Intracellular Ca2+ concentration and Ca2+ flux rate from the extracellular compartment was not changed by zoledronate administration. No changes in Rab27a/Doc2a interaction after zoledronate treatment. Histamine release inhibition by zoledronate in DNP-BSA-activated RBL-2H3 cells is not related to the disruption of Rab27a/Doc2a interaction and is not involve the change in Ca2+ influx.

Interleukin-6-interleukin-11 receptor chimeras reveal ionomycin-induced proteolysis beyond ADAM10.

Interleukin-6 (IL-6) and interleukin-11 (IL-11) are two important pleiotropic cytokines, both of which signal through a homodimer of the ß-receptor gp130. Specificity is gained through the unique, nonsignaling α-receptors IL-6R and IL-11R. Soluble variants of IL-6R and IL-11R also exist. Both membrane-bound receptors can be cleaved by the metalloprotease ADAM10. Here, we use ten different chimeric receptors consisting of different parts of IL-6R and IL-11R and analyze their susceptibility toward cleavage by ADAM10. As expected, all chimeras are substrates of ADAM10. However, we observed that cleavage of chimeric receptors containing the stalk region of the IL-11R could be blocked by the protease inhibitor GI (selective for ADAM10), but not by the protease inhibitor GW (selective for both ADAM10 and ADAM17), suggesting that another protease besides ADAM10 is involved in cleavage of these chimeras.

Low-intensity ultrasound (LIUS) differentially modulates mitochondrial reactive oxygen species (mtROS) generation by three different chemicals in PC12 cells.

We have previously shown that low-intensity ultrasound (LIUS) can modulate mitochondrial complex I activity and the generation of mitochondrial reactive oxygen species (mtROS) in PC12 cells. This study investigated the mechanism of LIUS by comparing its effect on mitochondrial dysfunction by three different pathways. LIUS was shown to reverse the effects of rotenone, a Q-site blocker, on the complex I inhibition, mtROS generation, and drop of mitochondrial membrane potential (Δψm). In contrast, common antioxidants, N-acetyl cysteine (NAC), and uric acid (UA) blocked rotenone-induced mtROS generation and Δψm drop without recovering the complex I activity, which suggested that Δψm drop is correlated with mtROS generation rather than complex I inhibition itself. Ionomycin, an ionophore for Ca2+, and L-buthionine-S,R-sulfoximine (BSO), an inhibitor of glutathione (GSH) biosynthesis, induced mtROS generation and Δψm drop without inhibiting complex I activity via different mechanisms. LIUS showed no effect on ionomycin-induced Δψm drop but showed partial inhibition on the other effects of ionomycin and BSO. These results suggest that LIUS might have redundant mechanisms but acted mainly on the complex I activity thereby modulating mtROS and Δψm levels. LIUS appeared to act on the Q-module of complex I because it showed no inhibitory effect on Zn2+, an inhibitor of the proton transporting P-module of complex I. Interestingly, pretreatment of LIUS for up to an hour in advance blocked the rotenone effect as efficiently as the co-treatment. Further studies are needed to reveal the exact mechanism of LIUS to inhibit complex I activity.