Inducible ATP1B1 Upregulates Antiviral Innate Immune Responses by the Ubiquitination of TRAF3 and TRAF6.

The antiviral innate immune responses are crucial steps during host defense and must be strictly regulated, but the molecular mechanisms of control remain unclear. In this study, we report increased expression of human ATPase Na+/K+ transporting subunit ß 1(ATP1B1) after DNA and RNA virus infections. We found that the expression of ATP1B1 can inhibit viral replication and increase the levels of IFNs, IFN-stimulated genes, and inflammatory cytokines. Knockdown of ATP1B1 by specific short hairpin RNA had the opposite effects. Upon viral infection, ATP1B1 was induced, interacted with TRAF3 and TRAF6, and potentiated the ubiquitination of these proteins, leading to increased phosphorylation of downstream molecules, including TGF-ß-activated kinase 1 (TAK1) and TANK-binding kinase 1 (TBK1). These results reveal a previously unrecognized role of ATP1B1 in antiviral innate immunity and suggest a novel mechanism for the induction of IFNs and proinflammatory cytokines during viral infection.

Osmoregulatory performance and immunolocalization of Na+/K+-ATPase in the branchiopod Artemia salina from the Sebkha of Sidi El Hani (Tunisia).

Artemia salina is an extremophile species that tolerates a wide range of salinity, especially hypertonic media considered lethal for the majority of other aquatic species. In this study, A. salina cysts were hatched in the laboratory and nauplii were acclimated at three different salinities (60, 139 and 212 ppt). Once in the adult phase, their hemolymph osmolality was measured. The animals were strong hypo-osmoregulators in the entire range of tested salinities, with up to 10 fold lower hemolymph osmolalities than their surrounding environment. Immunostaining of Na+/K+-ATPase was done on sections and on whole body mounts of adults in order to localize the ionocytes in different organs. An intense Na+/K+-ATPase immunostaining throughout the cells was observed in the epithelium of the ten pairs of metepipodites. A positive immunoreactivity for Na+/K+-ATPase was also detected in the maxillary glands, in the epithelium of the efferent tubule and of the excretory canal, as well as in the anterior digestive tract. This study confirms the strong hypo-osmotic capacity of this species and affords an overview of the different organs involved in osmoregulation in A. salina adults.

Immunization with Na+/K+ ATPase DR peptide prevents bone loss in an ovariectomized rat osteoporosis model.

Osteoporosis is characterized by decreased bone strength and microarchitectural deterioration of bone tissue leading to an increase in bone fracture. Here we report a new agent named DR peptide, a conserved sequence of Na+/K+ ATPase (NKA), can be used to prevent osteoporosis. Our results showed that immunization with DR peptide conjunct with Keyhole limpet hemocyanin (DR-KLH) significantly strengthened trabecular bone and improved bone mineral density of femur and the 5th lumbar in the ovariectomized (OVX) rats when compared with those in OVX rats immunized with KLH alone. To study the underlying mechanisms, anti-DR antibody (DR-Ab) and IgG were purified from the serum of rats immunized with DR-KLH or KLH alone in OVX rats. We found that DR-Ab had no significant effects on the proliferation of osteoclasts precursors, but it alleviated the inhibitory effect of H2O2 on the expression of osteogenic marker genes, Runx2, alkaline phosphatase and bone sialoprotein. DR-Ab also reversed the loss of collagen and the reduced bone nodule formation caused by H2O2 treatment. Knockdown of NKA with siRNA transfection reduced osteoblast differentiation and rendered the osteoblasts more vulnerable to oxidative challenge. Immunostaining analysis confirmed that NKA expression was reduced in osteoblasts in the femur of OVX rats, but this effect was attenuated by immunization with DR-KLH. Therefore, the protective effects of DR-Ab on osteoblasts might be associated with preservation of NKA level. Taken together, our findings reveal a novel mechanism for the development of osteoporosis and DR peptide and DR-Ab are potential approaches for the treatment of osteoporosis.

Ligation of Na, K ATPase ß3 subunit on monocytes by a specific monoclonal antibody mediates T cell hypofunction.

T cells play a crucial role in orchestrating body immune responses. T cell hyperfunction, however, leads to inflammation and induction of autoimmune diseases. Understanding of T cell regulation mechanisms and successful modulation of T cell responses is beneficial in treatment of disease associated to T cell hyperresponsiveness. Our previous study indicated that monoclonal antibody (mAb) P-3E10, a mAb to Na, K ATPase ß3 subunit, inhibited anti-CD3-induced PBMC proliferation. In the current study, we further investigated the mechanism of mAb P-3E10 in the induction of T cell hypofunction. We demonstrated that mAb P-3E10 decreased T cell proliferation and Th1, Th2 and Th17 cytokine production. Monocytes were the cells playing a key role in mediation of mAb P-3E10 induced T cell hypofunction. The inhibition of T cell activation by mAb P-3E10 required cell contact between monocytes and T cells. The mAb P-3E10 induced the down-expression level of MHC class II and CD86 and increased IL-6, IL-10 and TNF-α production of monocytes. We concluded that ligation of the Na, K ATPase ß3 subunit on monocytes by mAb P-3E10 arbitrated T cell hypofunction. This mAb might be a promising novel immunotherapeutic antibody for the treatment of hyperresponsive T cell associated diseases.

Na+, K+-ATPase Activating Antibody Displays in vitro and in vivo Beneficial Effects in the Pilocarpine Model of Epilepsy.

Na+, K+-ATPase is an important regulator of brain excitability. Accordingly, compelling evidence indicates that impairment of Na+, K+-ATPase activity contributes to seizure activity in epileptic mice and human with epilepsy. In addition, this enzyme is crucial for plasma membrane transport of water, glucose and several chemical mediators, including glutamate, the major excitatory transmitter in the mammalian brain. Since glucose hypometabolism and increased glutamate levels occur in clinical and experimental epilepsy, we aimed the present study to investigate whether activation of Na+, K+-ATPase activity with specific antibody (DRRSAb) would improve glucose uptake and glutamate release in pilocarpine-treated mice. We found decreased uptake of the glucose fluorescent analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-il)amino]-2-desoxi-d-glucose (2-NBDG) in cerebral slices from pilocarpine-treated animals. Interestingly, decreased 2-NBDG uptake was not detected in DRRSAb-treated slices, suggesting a protective effect of the Na+, K+-ATPase activator. Moreover, DRRSAb prevented the increase in glutamate levels in the incubation media of slices from pilocarpine-treated mice. In addition, in vivo intrahippocampal injection of DRRSAb restored crossing activity of pilocarpine-treated mice in the open-field test. Overall, the present data further support the hypothesis that activation of the Na+, K+-ATPase is a promising therapeutic strategy for epilepsy.

Cerebral Malaria Causes Enduring Behavioral and Molecular Changes in Mice Brain Without Causing Gross Histopathological Damage.

Malaria, parasitic disease considered a major health public problem, is caused by Plasmodium protozoan genus and transmitted by the bite of infected female Anopheles mosquito genus. Cerebral malaria (CM) is the most severe presentation of malaria, caused by P. falciparum and responsible for high mortality and enduring development of cognitive deficits which may persist even after cure and cessation of therapy. In the present study we evaluated selected behavioral, neurochemical and neuropathologic parameters after rescue from experimental cerebral malaria caused by P. berghei ANKA in C57BL/6 mice. Behavioral tests showed impaired nest building activity as well as increased marble burying, indicating that natural behavior of mice remains altered even after cure of infection. Regarding the neurochemical data, we found decreased α2/α3 Na+,K+-ATPase activity and increased immunoreactivity of phosphorylated Na+,K+-ATPase at Ser943 in cerebral cortex after CM. In addition, [3H]-Flunitrazepam binding assays revealed a decrease of benzodiazepine/GABAA receptor binding sites in infected animals. Moreover, in hippocampus, dot blot analysis revealed increased levels of protein carbonyls, suggesting occurrence of oxidative damage to proteins. Interestingly, no changes in the neuropathological markers Fluoro-Jade C, Timm staining or IBA-1 were detected. Altogether, present data indicate that behavioral and neurochemical alterations persist even after parasitemia clearance and CM recovery, which agrees with available clinical findings. Some of the molecular mechanisms reported in the present study may underlie the behavioral changes and increased seizure susceptibility that persist after recovery from CM and may help in the future development of therapeutic strategies for CM sequelae.

Anticancer and Immunogenic Properties of Cardiac Glycosides.

Cardiac glycosides (CGs) are natural compounds widely used in the treatment of several cardiac conditions and more recently have been recognized as potential antitumor compounds. They are known to be ligands for Na/K-ATPase, which is a promising drug target in cancer. More recently, in addition to their antitumor effects, it has been suggested that CGs activate tumor-specific immune responses. This review summarizes the anticancer aspects of CGs as new strategies for immunotherapy and drug repositioning (new horizons for old players), and the possible new targets for CGs in cancer cells.

Activation of Na+-K+-ATPase with DRm217 attenuates oxidative stress-induced myocardial cell injury via closing Na+-K+-ATPase/Src/Ros amplifier.

Reduced Na+-K+-ATPase activity has close relationship with cardiomyocyte death. Reactive oxygen species (ROS) also plays an important role in cardiac cell damage. It has been proved that Na+-K+-ATPase and ROS form a feed-forward amplifier. The aim of this study was to explore whether DRm217, a proved Na+/K+-ATPase's DR-region specific monoclonal antibody and direct activator, could disrupt Na+-K+-ATPase/ROS amplifier and protect cardiac cells from ROS-induced injury. We found that DRm217 protected myocardial cells against hydrogen peroxide (H2O2)-induced cardiac cell injury and mitochondrial dysfunction. DRm217 also alleviated the effect of H2O2 on inhibition of Na+-K+-ATPase activity, Na+-K+-ATPase cell surface expression, and Src phosphorylation. H2O2-treatment increased intracellular ROS, mitochondrial ROS and induced intracellular Ca2+, mitochondrial Ca2+ overload. DRm217 closed Na+-K+-ATPase/ROS amplifier, alleviated Ca2+ accumulation and finally inhibited ROS and mitochondrial ROS generation. These novel results may help us to understand the important role of the Na+-K+-ATPase in oxidative stress and oxidative stress-related disease.

[Preparation and application of monoclonal antibodies against DR region of Na+-K+-ATPase α1 subunit].

Objective To prepare monoclonal antibodies against DR region (897DVEDSYGQQWTYEQR911) of Na+-K+-ATPase α1 subunit and identify their properties. Methods BALB/c mice were immunized with DR-keyholelimpet hemocyanin (KLH). Splenocytes from the immunized mice were collected and subsequently fused with SP2/0 mouse myeloma cells. Positive hybridoma clones were obtained after cell fusion and selection. ELISA was used to detect DR antibody titer in the cell supernatants. DR region-specific monoclonal antibodies were analyzed by dot blotting, Western blotting and immunofluorescence assay. Na+-K+-ATPase activity was detected by SensoLyteR FDP Protein Phosphatase Assay Kit and the protective effect of the monoclonal antibody against high glucose-induced cell injury was assessed in H9c2 cells. Results Three hybridoma cell lines which secreted stable DR monoclonal antibody were obtained. The strongest positive cell line, named DRm217, was selected to prepare ascites. Dot blotting, Western blotting and immunofluorescence assay showed that DRm217 recognized specially DR region of Na+-K+-ATPase and bound on H9c2 cell membranes. DRm217 stimulated Na+-K+-ATPase activity and alleviated high glucose-induced H9c2 cells injury. Conclusion The monoclonal antibodies against DR region of Na+-K+-ATPase α1 subunit is prepared.

Activation of Na+/K+-ATPase attenuates high glucose-induced H9c2 cell apoptosis via suppressing ROS accumulation and MAPKs activities by DRm217.

Hyperglycemia is one of the major factors responsible for the myocardial apoptosis and dysfunction in diabetes. Many studies have proved that there is a close relationship between decreased Na+/K+-ATPase activity and diabetic cardiomyopathy. However, the effect of directly activated Na+/K+-ATPase on high glucose-induced myocardial injury is still unknown. Here we found that DRm217, a Na+/K+-ATPase's DR-region specific monoclonal antibody and direct activator, could prevent high glucose-induced H9c2 cell injury, reactive oxygen species (ROS) release, and mitochondrial dysfunction. High glucose-treatment decreased Na+/K+-ATPase activity and increased intracellular Ca2+ level, whereas DRm217 increased Na+/K+-ATPase activity and alleviated Ca2+ overload. Inhibition of Ca2+ overload or closing sodium calcium exchanger (NCX channel) could reverse high glucose-induced ROS increasing and cell injury. In addition, DRm217 could significantly attenuate high glucose-induced p38, JNK and ERK1/2 phosphorylation, which were involved in high glucose-induced cell injury and ROS accumulation. Our findings suggest that DRm217 may protect against the deleterious effects of high glucose in the heart. Prevention of high glucose-induced myocardial cell injury by specific Na+/K+-ATPase activator may be an attractive therapeutic option.