SUMMARY Ca2+ is an essential and ubiquitous second messenger. Changes in cytosolic Ca2+ trigger events critical for tumorigenesis, such as cellular motility, proliferation and apoptosis. We show that an isoform of Secretory Pathway Ca2+-ATPase, SPCA2, is upregulated in breast cancer-derived cells and human breast tumors, and suppression of SPCA2 attenuated basal Ca2+ levels and tumorigenicity. Contrary to its conventional role in Golgi Ca2+ sequestration, expression of SPCA2 increased Ca2+ influx by a mechanism dependent on the store-operated Ca2+ channel Orai1. Unexpectedly, SPCA2-Orai1 signaling was independent of ER Ca2+ stores or STIM1 and STIM2 sensors, and uncoupled from Ca2+-ATPase activity of SPCA2. Binding of SPCA2 amino terminus to Orai1 enabled access of its carboxyl terminus to Orai1 and activation of Ca2+ influx. Our findings reveal a signaling pathway in which Orai1-SPCA2 complex elicits constitutive store-independent Ca2+ signaling that promotes tumorigenesis.

Terpenoid phenols, including carvacrol, are components of oregano and other plant essential oils that exhibit potent antifungal activity against a wide range of pathogens, including Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa. To gain a mechanistic view of the cellular response to terpenoid phenols, we used Saccharomyces cerevisiae as a model organism and monitored temporal changes in metabolic activity, cytosolic and vacuolar pH, and Ca 2؉ transients. Using a panel of related compounds, we observed dosedependent Ca 2؉ bursts that correlated with antifungal efficacy. Changes in pH were long lasting and followed the Ca 2؉ transients. A vma mutant lacking functional vacuolar H ؉ -ATPase (V-ATPase) and defective in ion homeostasis was hypersensitive to carvacrol toxicity, consistent with a role for ionic disruptions in mediating cell death. Genomic profiling within 15 min of exposure revealed a robust transcriptional response to carvacrol, closely resembling that of calcium stress. Genes involved in alternate metabolic and energy pathways, stress response, autophagy, and drug efflux were prominently upregulated, whereas repressed genes mediated ribosome biogenesis and RNA metabolism. These responses were strongly reminiscent of the effects of rapamycin, the inhibitor of the TOR pathway of nutrient sensing. The results point to the activation of specific signaling pathways downstream of cellular interaction with carvacrol rather than a nonspecific lesion of membranes, as has been previously proposed.While the medicinal properties of herbs have been recognized since ancient times, there has been a resurgence of interest in the antimicrobial properties of botanical extracts. Essential oils have been amply documented to kill a wide range of pathogenic fungi and bacteria, such as Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa, including their drug-resistant variants (6,10,21,22). Of the herbal extracts tested, essential oils derived from the genus Oreganum were among the most effective, with an in vitro MIC of 500 ppm against C. albicans (27). Major components of oregano extract, which include the terpenoid phenols carvacrol, thymol, and eugenol, have potent antifungal activity of their own (4, 23, 24). Terpenoid phenols have been shown to be efficacious not only on planktonic cells but also on biofilms of Candida albicans that are resistant to many antifungal drugs. Carvacrol demonstrated the strongest antifungal activity against Candida albicans biofilms, with a MIC of Ͻ0.03% (9). Furthermore, carvacrol was shown to be effective regardless of the maturity of the biofilm. The terpenoid phenols tested were able to inhibit biofilms of several strains of Candida, including C. albicans, C. glabrata, and C. parapsilosis. In addition to their antimycotic, antibacterial, insecticidal, and bioherbicidal properties, essential oils are also well known for their antioxidant characteristics and are used to inhibit lipid peroxidation in preventing food spoilage or as chemoprotective agen...

Phylogenetic analysis of the cation/proton antiporter superfamily has uncovered a previously unknown clade of genes in metazoan genomes, including two previously uncharacterized human isoforms, NHA1 and NHA2, found in tandem on human chromosome 4. The NHA (sodium hydrogen antiporter) family members share significant sequence similarity with Escherichia coli NhaA, including a conserved double aspartate motif in predicted transmembrane 5. We show that HsNHA2 (Homo sapiens NHA2) resides on the plasma membrane and, in polarized MDCK cells, localizes to the apical domain. Analysis of mouse tissues indicates that NHA2 is ubiquitous. When expressed in the yeast Saccharomyces cerevisiae lacking endogenous cation/proton antiporters and pumps, HsNHA2 can confer tolerance to Li ؉ and Na ؉ ions but not to K ؉ . HsNHA2 transformants accumulated less Li ؉ than the salt-sensitive host; however, mutagenic replacement of the conserved aspartates abolished all observed phenotypes. Functional complementation by HsNHA2 was insensitive to amiloride, a characteristic inhibitor of plasma membrane sodium hydrogen exchanger isoforms, but was inhibited by phloretin. These are hallmarks of sodium-lithium countertransport activity, a highly heritable trait correlating with hypertension. Our findings raise the possibility that NHA genes may contribute to sodium-lithium countertransport activity and salt homeostasis in humans.sodium-lithium countertransport ͉ yeast expression ͉ red blood cell ͉ pancreas