Dynamic interaction of SARAF with STIM1 and Orai1 to modulate store-operated calcium entry.

Ca(2+) influx by store-operated Ca(2+) channels is a major mechanism for intracellular Ca(2+) homeostasis and cellular function. Here we present evidence for the dynamic interaction between the SOCE-associated regulatory factor (SARAF), STIM1 and Orai1. SARAF overexpression attenuated SOCE and the STIM1-Orai1 interaction in cells endogenously expressing STIM1 and Orai1 while RNAi-mediated SARAF silencing induced opposite effects. SARAF impaired the association between Orai1 and the Orai1-activating small fragment of STIM1 co-expressed in the STIM1-deficient NG115-401L cells. Cell treatment with thapsigargin or physiological agonists results in direct association of SARAF with Orai1. STIM1-independent interaction of SARAF with Orai1 leads to activation of this channel. In cells endogenously expressing STIM1 and Orai1, Ca(2+) store depletion leads to dissociation of SARAF with STIM1 approximately 30s after treatment with thapsigargin, which paralleled the increase in SARAF-Orai1 interaction, followed by reinteraction with STIM1 and dissociation from Orai1. Co-expression of SARAF and either Orai1 or various N-terminal deletion Orai1 mutants did not alter SARAF-Orai1 interaction; however, expression of C-terminal deletion Orai1 mutants or blockade of the C-terminus of Orai1 impair the interaction with SARAF. These observations suggest that SARAF exerts an initial positive role in the activation of SOCE followed by the facilitation of SCDI of Orai1.

TNF-alpha and its inhibitors in cancer.

Tumor necrosis factor (TNF)-alpha is implicated in the same time in apoptosis and in cell proliferation. TNF-alpha not only acts as pro-inflammatory cytokine conducing to wide spectrum of human diseases including inflammatory diseases, but can also induce tumor development. The molecular mechanisms of TNF-alpha functions have been intensively investigated. In this review we covered TNF-alpha, the molecule, its signaling pathway, and its therapeutic functions. We provide a particular insight in its paradoxical role in tumor promotion and in its use as anti-tumor agent. This review considers also the recent findings regarding TNF-alpha inhibitors, their pharmacokinetics, and their pharmacodynamics. Six TNF-alpha inhibitors have been considered here: Infliximab, Adalimumab, Golimumab, CDP870, CDP571, Etanercept, and Thalidomide. We discussed the clinical relevance of their functions in treatment of several diseases such as advanced inflammatory rheumatic and bowel disease, with a focus in cancer treatment. Targeting TNF-alpha by these drugs has many side effects like malignancies development, and the long-term sequels are not very well explored. Their efficacy and their safety were discussed, underscoring the necessity of close patients monitoring and of their caution use.

Acidic-store depletion is required for human platelet aggregation.

Platelet stimulation with thrombin induces an elevation in cytoplasmic free Ca(2+) concentration ([Ca(2+)]c) due to Ca(2+) release from intracellular stores and entry from the extracellular medium. Two different intracellular Ca(2+) stores have been described in human platelets: the dense tubular system and the lysosomal-like acidic stores. In the present study, we investigated the contribution of the acidic stores in thrombin-induced platelet aggregation. We have found that platelet aggregation induced by thrombin is reduced in a Ca(2+)-free medium. Discharge of the acidic Ca(2+) stores by treatment with the sarcoendoplasmic Ca(2+)-ATPase (SERCA)3 selective inhibitor 2,5-di-(tert-butyl)-1,4-hydroquinone reduced thrombin-evoked platelet aggregation. In the presence of 2,5-di-(tert-butyl)-1,4-hydroquinone, platelet aggregation induced by the protease-activated receptor (PAR)-1 and PAR-4 agonist peptides, SFLLRN and AYPGKF, respectively, was significantly reduced. In cells with depleted acidic stores, activation of GPIb-IX-V by thrombin resulted in reduced or no platelet aggregation in a medium containing 1 mmol/l Caor in a Ca(2+)-free medium, respectively. This finding suggests that Ca(2+) accumulation in the acidic Ca(2+) compartments is required for platelet aggregation induced by activation of the G-coupled PAR-1 and PAR-4 thrombin receptors and, by the occupation of the leucine-rich glycoprotein GPIb-IX-V and provide evidence supporting a functional role of the lysosomal-like acidic Ca(2+) stores in human platelets.

Enhanced expression of STIM1/Orai1 and TRPC3 in platelets from patients with type 2 diabetes mellitus.

Type 2 diabetes mellitus (DM2) is a metabolic syndrome that contributes to both macrovascular and microvascular disorders, where platelet hyperaggregability, associated to abnormal intracellular Ca(2+) homeostasis, plays an important role. We have now investigated the expression of different proteins associated to Ca(2+) entry, a major Ca(2+) signalling event. DM2 donors were randomly selected from normotensive patients with glycosylated Hb levels (HbA1c) over 6%. Control subjects were normal age- and gender-matched healthy people with HbA1c levels in the normal range (3.5-5%). Expression of TRPC1, 3 and 6, STIM1 and Orai1 was analyzed by Western blotting in DM2 patients and controls. Expression of TRPC1 in platelets from DM2 donors and controls was similar; however, expression of TRPC6 is reduced in platelets from DM2 patients as compared to healthy controls. We have found that expression of TRPC3, Orai1 and STIM1 is enhanced in DM2 subjects as compared to controls. Our findings provide an explanation to the enhanced Ca(2+) entry induced by physiological agonists in platelets from DM2 patients.

Tyrosine phosphorylation / dephosphorylation balance is involved in thrombin-evoked microtubular reorganisation in human platelets.

We have investigated the intracellular mechanisms involved in microtubular remodelling by thrombin and its possible involvement in platelet aggregation and secretion. Platelet stimulation with thrombin induces a time- and concentration-dependent regulation of the microtubular content, which was found to be maximally effective at the concentration 0.1 U/ml. Thrombin (0.1 U/ml) evoked an initial decrease in the microtubule content detectable at 5 seconds (sec) and reached a minimum 10 sec after stimulation. The microtubular content then increased, exceeding basal levels again approximately 30 sec after stimulation. Inhibition of tyrosine phosphatases using vanadate abolished thrombin-induced microtubular depolymerisation while inhibition of tyrosine kinases by methyl-2,5-dihydroxycinnamate prevented microtubule polymerisation. Thrombin activates the cytosolic Bruton's tyrosine kinase (Btk) and Src proteins. Inhibition of Btk or Src by LFM-A13 or PP1, respectively, abolished thrombin-induced microtubular polymerisation, while maintaining intact its ability to induce initial depolymerisation. Microtubular disruption by colchicine significantly reduced thrombin-induced platelet aggregation and ATP secretion. Similar results were observed after inhibition of microtubular disassembly by paclitaxel. These findings indicate that thrombin induces microtubular remodelling by modifying the balance between protein tyrosine phosphorylation and dephosphorylation. The former seems to be required for microtubular polymerisation, while tyrosine dephosphorylation is required for microtubular depolymerisation. Both, initial microtubular disassembly and subsequent polymerisation are required for thrombin-induced platelet aggregation and secretion in human platelets.