No abstract

Orai1 and hTRPC1 have been presented as essential components of store-operated channels mediating highly Ca(2+) selective I(CRAC) and relatively Ca(2+) selective I(SOC), respectively. STIM1 has been proposed to communicate the Ca(2+) content of the intracellular Ca(2+) stores to the plasma membrane store-operated Ca(2+) channels. Here we present evidence for the dynamic interaction between endogenously expressed Orai1 and both STIM1 and hTRPC1 regulated by depletion of the intracellular Ca(2+) stores, using the pharmacological tools thapsigargin plus ionomycin, or by the physiological agonist thrombin, independently of extracellular Ca(2+). In addition we report that Orai1 mediates the communication between STIM1 and hTRPC1, which is essential for the mode of activation of hTRPC1-forming Ca(2+) permeable channels. Electrotransjection of cells with anti-Orai1 antibody, directed toward the C-terminal region that mediates the interaction with STIM1, and stabilization of an actin cortical barrier with jasplakinolide prevented the interaction between STIM1 and hTRPC1. Under these conditions hTRPC1 was no longer involved in store-operated calcium entry but in diacylglycerol-activated non-capacitative Ca(2+) entry. These findings support the functional role of the STIM1-Orai1-hTRPC1 complex in the activation of store-operated Ca(2+) entry.

To cite this article: Lopez JJ, Salido GM, Gó mez-Arteta E, Rosado JA, Pariente JA. Thrombin induces apoptotic events through the generation of reactive oxygen species in human platelets. J Thromb Haemost 2007; 5: 1283-91.Summary. Background: Thrombin is a major physiological platelet agonist that activates a number of cell functions including aggregation. Platelet stimulation with thrombin has been shown to result in the development of apoptotic events, including activation of caspases-3 and -9, cytochrome c release and phosphatidylserine (PS) exposure; however, the mechanism underlying the activation of apoptosis remains unclear. Objectives: In the present study, we aim to investigate whether endogenously generated reactive oxygen species upon thrombin stimulation is required for the activation of apoptosis in human platelets. Methods: Changes in the mitochondrial membrane potential were registered using the dye JC-1; caspase-3 and -9 activity was determined from the cleavage of their respective specific fluorogenic substrates; PS externalization was estimated using annexin V-fluorescein isothicyanate and cytochrome c release was detected by Western blotting in samples from the mitochondrial and cytosolic fractions. Results: Treatment of platelets with thrombin stimulates mitochondrial membrane potential depolarization and endogenous generation of H 2 O 2 . Platelet exposure to exogenous H 2 O 2 results in cytochrome c release and activation of caspases-9. In addition, H 2 O 2 induces the activation of caspase-3 and PS exposure by a mechanism dependent on cytochrome c release and caspase-9 activation. Finally, thrombin-evoked development of apoptotic events was impaired by treatment with catalase. Conclusion: Our results indicate that thrombininduced apoptosis is likely mediated by endogenous generation of H 2 O 2 in human platelets.

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.

Transient receptor potential channels convey signaling information from a number of stimuli to a wide variety of cellular functions, mainly by inducing changes in cytosolic Ca2+ concentration. Different members of the TRPC, TRPM and TRPV subfamilies have been reported to play a role in tumorigenesis. Here we show that the estrogen receptor positive and triple negative breast cancer cell lines, MCF7 and MDA-MB-231, respectively, exhibit enhanced expression of the TRPC6 channel as compared to the non-tumoral MCF10A cell line. In vitro TRPC6 knockdown using shRNA impaired MCF7 and MDA-MB-231 cell proliferation, migration and invasion detected by BrdU incorporation, wound healing and Boyden chamber assays, respectively. Using RNAi-mediated TRPC6 silencing as well as overexpression of the pore-dead dominant-negative TRPC6 mutant we have found that TRPC6 plays a relevant role in the activation of store-operated Ca2+ entry in the breast cancer cell lines but not in non-tumoral breast cells. Finally, we have found that TRPC6 interacts with Orai1 and Orai3 in MCF7 and MDA-MB-231 cells and is required for the translocation of Orai1 and Orai3 to the plasma membrane in MDA-MB-231 and MCF7 cells, respectively, upon Ca2+ store depletion. These findings introduce a novel mechanism for the modulation of Ca2+ influx and the development of different cancer hallmarks in breast cancer cells.