Essential role of Orai1 store-operated calcium channels in lactation.

The nourishment of neonates by nursing is the defining characteristic of mammals. However, despite considerable research into the neural control of lactation, an understanding of the signaling mechanisms underlying the production and expulsion of milk by mammary epithelial cells during lactation remains largely unknown. Here we demonstrate that a store-operated Ca(2+) channel subunit, Orai1, is required for both optimal Ca(2+) transport into milk and for milk ejection. Using a novel, 3D imaging strategy, we visualized live oxytocin-induced alveolar unit contractions in the mammary gland, and we demonstrated that in this model milk is ejected by way of pulsatile contractions of these alveolar units. In mammary glands of Orai1 knockout mice, these contractions are infrequent and poorly coordinated. We reveal that oxytocin also induces a large transient release of stored Ca(2+) in mammary myoepithelial cells followed by slow, irregular Ca(2+) oscillations. These oscillations, and not the initial Ca(2+) transient, are mediated exclusively by Orai1 and are absolutely required for milk ejection and pup survival, an observation that redefines the signaling processes responsible for milk ejection. These findings clearly demonstrate that Ca(2+) is not just a substrate for nutritional enrichment in mammals but is also a master regulator of the spatiotemporal signaling events underpinning mammary alveolar unit contraction. Orai1-dependent Ca(2+) oscillations may represent a conserved language in myoepithelial cells of other secretory epithelia, such as sweat glands, potentially shedding light on other Orai1 channelopathies, including anhidrosis (an inability to sweat).

Role of the store-operated calcium entry protein, STIM1, in neutrophil chemotaxis and infiltration into a murine model of psoriasis-inflamed skin.

Stromal interaction molecule 1 (STIM1) is a Ca(2+) sensor protein that initiates store-operated calcium entry (SOCE). STIM1 is known to be involved in the chemoattractant signaling pathway for FPR1 in cell lines, but its role in in vivo functioning of neutrophils is unclear. Plaque-type psoriasis is a chronic inflammatory skin disorder associated with chemoattractants driving neutrophils into the epidermis. We investigated the involvement of STIM1 in neutrophil chemotaxis in vitro, as well as during chronic psoriatic inflammation. To this end, we used conditional knockout (KO) mice lacking STIM1 in cells of myeloid lineage (STIM1(fl/fl) LysM-cre). We demonstrate that STIM1 is required for chemotaxis because of multiple chemoattractants in mouse neutrophils in vitro. Using an imiquimod-induced psoriasis-like skin model, we show that KO mice had less neutrophil infiltration in the epidermis than controls, whereas neither chemoattractant production in the epidermis nor macrophage migration was decreased. KO mice displayed a more rapid reversal of the outward signs of psoriasis (plaques). Thus, KO of STIM1 impairs neutrophil contribution to psoriatic inflammation. Our data provide new insights to our understanding of how STIM1 orchestrates the cellular behavior underlying chemotaxis and illustrate the important role of SOCE in a disease-related pathologic model.

Role of Orai1 and store-operated calcium entry in mouse lacrimal gland signalling and function.

Lacrimal glands function to produce an aqueous layer, or tear film, that helps to nourish and protect the ocular surface. Lacrimal glands secrete proteins, electrolytes and water, and loss of gland function can result in tear film disorders such as dry eye syndrome, a widely encountered and debilitating disease in ageing populations. To combat these disorders, understanding the underlying molecular signalling processes that control lacrimal gland function will give insight into corrective therapeutic approaches. Previously, in single lacrimal cells isolated from lacrimal glands, we demonstrated that muscarinic receptor activation stimulates a phospholipase C-coupled signalling cascade involving the inositol trisphosphate-dependent mobilization of intracellular calcium and the subsequent activation of store-operated calcium entry (SOCE). Since intracellular calcium stores are finite and readily exhausted, the SOCE pathway is a critical process for sustaining and maintaining receptor-activated signalling. Recent studies have identified the Orai family proteins as critical components of the SOCE channel activity in a wide variety of cell types. In this study we characterize the role of Orai1 in the function of lacrimal glands using a mouse model in which the gene for the calcium entry channel protein, Orai1, has been deleted. Our data demonstrate that lacrimal acinar cells lacking Orai1 do not exhibit SOCE following activation of the muscarinic receptor. In comparison with wild-type and heterozygous littermates, Orai1 knockout mice showed a significant reduction in the stimulated tear production following injection of pilocarpine, a muscarinic receptor agonist. In addition, calcium-dependent, but not calcium-independent exocytotic secretion of peroxidase was eliminated in glands from knockout mice. These studies indicate a critical role for Orai1-mediated SOCE in lacrimal gland signalling and function.

Deletion of Orai1 alters expression of multiple genes during osteoclast and osteoblast maturation.

Store-operated Ca(2+) entry (SOCE) is a major Ca(2+) influx pathway in most non-excitable cell types and Orai1 was recently identified as an essential pore-subunit of SOCE channels. Here we investigate the physiological role of Orai1 in bone homeostasis using Orai1-deficient mice (Orai1(-/-)). Orai1(-/-) mice developed osteopenia with decreased bone mineral density and trabecular bone volume. To identify the nature and origin of the bone defect, bone-resorbing osteoclasts and bone-forming osteoblasts from Orai1(-/-) mice were examined. Orai1-mediated SOCE was completely abolished in Orai1(-/-) osteoclast precursor cells and osteoclastogenesis in vitro from Orai1(-/-) mice was impaired due to a defect in cell fusion of pre-osteoclasts. Also, resorption activity in vitro was comparable but the size of pits formed by Orai1(-/-) osteoclasts was smaller. We next assessed the role of Orai1 in osteoblast differentiation and function by using a pre-osteoblast cell line, as well as primary osteoblasts from wild-type and Orai1(-/-) mice. SOCE in MC3T3-E1 pre-osteoblastic cells was inactivated by lentiviral overexpression of a pore-dead Orai1 mutant. Lack of SOCE in MC3T3-E1 had no effect on alkaline phosphatase staining and expression but substantially inhibited mineralized nodule formation. Consistent with this finding, Orai1-mediated SOCE was markedly reduced in Orai1(-/-) osteoblast precursor cells and osteoblastogenesis in vitro from Orai1(-/-) stromal cells showed impaired mineral deposition but no change in differentiation. This indicates that Orai1 is involved in the function but not in the differentiation of osteoblasts. Together, these results suggest that Orai1 plays a critical role in bone homeostasis by regulating both osteoblasts and osteoclasts.

Phosphorylation of STIM1 underlies suppression of store-operated calcium entry during mitosis.

Store-operated Ca(2+) entry (SOCE) and Ca(2+) release-activated Ca(2+) currents (I(crac)) are strongly suppressed during cell division, the only known physiological situation in which Ca(2+) store depletion is uncoupled from the activation of Ca(2+) influx [corrected]. We found that the endoplasmic reticulum (ER) Ca(2+) sensor STIM1 failed to rearrange into near-plasma membrane puncta in mitotic cells, a critical step in the SOCE-activation pathway. We also found that STIM1 from mitotic cells is recognized by the phospho-specific MPM-2 antibody, suggesting that STIM1 is phosphorylated during mitosis. Removal of ten MPM-2 recognition sites by truncation at amino acid 482 abolished MPM-2 recognition of mitotic STIM1, and significantly rescued STIM1 rearrangement and SOCE response in mitosis. We identified Ser 486 and Ser 668 as mitosis-specific phosphorylation sites, and STIM1 containing mutations of these sites to alanine also significantly rescued mitotic SOCE. Therefore, phosphorylation of STIM1 at Ser 486 and Ser 668, and possibly other sites, underlies suppression of SOCE during mitosis.

TRPC channels function independently of STIM1 and Orai1.

Recent studies have defined roles for STIM1 and Orai1 as calcium sensor and calcium channel, respectively, for Ca(2+)-release activated Ca(2+) (CRAC) channels, channels underlying store-operated Ca(2+) entry (SOCE). In addition, these proteins have been suggested to function in signalling and constructing other channels with biophysical properties distinct from the CRAC channels. Using the human kidney cell line, HEK293, we examined the hypothesis that STIM1 can interact with and regulate members of a family of non-selective cation channels (TRPC) which have been suggested to also function in SOCE pathways under certain conditions. Our data reveal no role for either STIM1 or Orai1 in signalling of TRPC channels. Specifically, Ca(2+) entry seen after carbachol treatment in cells transiently expressing TRPC1, TRPC3, TRPC5 or TRPC6 was not enhanced by the co-expression of STIM1. Further, knockdown of STIM1 in cells expressing TRPC5 did not reduce TRPC5 activity, in contrast to one published report. We previously reported in stable TRPC7 cells a Ca(2+) entry which was dependent on TRPC7 and appeared store-operated. However, we show here that this TRPC7-mediated entry was also not dependent on either STIM1 or Orai1, as determined by RNA interference (RNAi) and expression of a constitutively active mutant of STIM1. Further, we determined that this entry was not actually store-operated, but instead TRPC7 activity which appears to be regulated by SERCA. Importantly, endogenous TRPC activity was also not regulated by STIM1. In vascular smooth muscle cells, arginine-vasopressin (AVP) activated non-selective cation currents associated with TRPC6 activity were not affected by RNAi knockdown of STIM1, while SOCE was largely inhibited. Finally, disruption of lipid rafts significantly attenuated TRPC3 activity, while having no effect on STIM1 localization or the development of I(CRAC). Also, STIM1 punctae were found to localize in regions distinct from lipid rafts. This suggests that TRPC signalling and STIM1/Orai1 signalling occur in distinct plasma membrane domains. Thus, TRPC channels appear to be activated by mechanisms dependent on phospholipase C which do not involve the Ca(2+) sensor, STIM1.

Ephedrine plus caffeine causes age-dependent cardiovascular responses in Fischer 344 rats.

Human consumption of ephedrine and caffeine in dietary supplements has been associated with a number of adverse effects including changes in the ECG, myocardial infarction, hyperthermia, and, in rare instances, death. The purpose of this study was to investigate the potential mechanisms associated with the cardiotoxicity of combined ephedrine and caffeine ingestion. Seven- and fourteen-week-old Fischer 344 rats treated with ephedrine in combination with caffeine exhibited increases in heart rate (HR), temperature, and corrected QT interval. Of the 14-wk-old rats treated with 25 mg/kg ephedrine plus 30 mg/kg caffeine, 57% died within 3-5 h of treatment, whereas none of the similarly treated 7-wk-old rats nor any of the rats treated with vehicle died. One hour after treatment with this dose of ephedrine plus caffeine, 14-wk-old rats exhibited a larger increase in HR (as % increase over baseline) than 7-wk-old rats. Furthermore, the 14-wk-old rats that died had a higher HR and temperature than the 14-wk-old rats that lived. Histopathological studies suggested interstitial hemorrhage and myofiber necrosis in the 14-wk-old rats treated with the highest concentration of ephedrine and caffeine. This study showed enhanced susceptibility to ephedrine plus caffeine in 14-wk-old rats compared with 7-wk-old rats. The greater mortality in the 14-wk-old rats was associated with increases in body temperature, HR, and myocardial necrosis.

Alterations in apoptotic signaling in human idiopathic cardiomyopathic hearts in failure.

Dilated cardiomyopathy, a disease of unknown etiology and pathogenesis, is associated with heart failure and compensatory hypertrophy. Although cell and animal models suggest a role for altered gene expression in the transition to heart failure, there is a paucity of data derived from the study of human heart tissue. In this study, we used DNA microarray profiling to investigate changes in the expression of genes involved in apoptosis that occur in human idiopathic dilated cardiomyopathic hearts that had progressed to heart failure. We observed altered gene expression consistent with a proapoptotic shift in the TNF-alpha signaling pathway. Specifically, we found decreased expression of TNF-alpha- and NF-kappaB-induced antiapoptotic genes such as growth arrest and DNA damage-inducible (GADD)45beta, Flice inhibitory protein (FLIP), and TNF-induced protein 3 (A20). Consistent with a role for apoptosis in heart failure, we also observed a significant decrease in phosphorylation of BAD at Ser-112. This study identifies several pathways that are altered in human heart failure and provides new targets for therapy.