John G. Petranka

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 Ca2+‐release activated Ca2+ (CRAC) channels, channels underlying store‐operated Ca2+ 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, Ca2+ 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 Ca2+ 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 ICRAC. 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 Ca2+ sensor, STIM1.

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

Store-operated Ca2+ entry (SOCE) and Ca2+ release-activated Ca2+ currents (Icrac) are strongly suppressed during cell division, the only known physiological situation in which Ca2+ store depletion is uncoupled from the activation of Ca2+ influx. We found that the endoplasmic reticulum (ER) Ca2+ 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.

The oncostatic action of melatonin in an ovarian carcinoma cell line

Abstract: Melatonin is reported to reduce proliferation in many cell types, but the effect is small and the results are inconsistent. Information on the mechanism by which melatonin exerts its antiproliferative effects might provide insight into the variability of the response. In an ovarian adenocarcinoma cell line (BG‐1), we find that melatonin at concentrations of 10−9‐10−7 M caused a 20–25% reduction in cell number. Melatonin also resulted in a similar reduction in [3H]‐thymidine incorporation with no significant increase in cell death as measured by trypan blue incorporation. The Kd for melatonin reduction in cell number was ∼ 5 × 10−10 M. Melatonin ML2 receptors have a Kd for melatonin binding in the low nM range and are linked to the production of the calcium mobilizing agent inositol‐1, 4, 5‐trisphosphate (IP3). To investigate whether melatonin signaling involves an increase in cytosolic‐free calcium, BG‐1 cells were loaded with the calcium sensitive indicator, fura‐2. Acute addition of melatonin (10−5‐10−9 M) did not alter cytosolic calcium. Addition of the putative nuclear receptor agonist CGP52608 caused a dose‐dependent inhibition of cell number with a Kd of ∼ 2 × 10−9 M. Addition of CGP52608 caused a similar reduction in [3H]‐thymidine incorporation. Neither melatonin (10−8 M‐10−5 M) nor CGP52608 at concentrations below 10−7 M induced cell death associated with the inhibition of cell proliferation; however, addition of CGP52608 at a high dose (10−7 M) caused an increase in cell death, consistent with apoptosis. Growth inhibition by melatonin or CGP52608 did not alter the percentage of cells in G1 versus S/G2/M.

REDUCED CAPACITATIVE CALCIUM ENTRY CORRELATES WITH VESICLE ACCUMULATION AND APOPTOSIS

A preneoplastic variant of Syrian hamster embryo cells, sup(+), exhibits decreased endoplasmic reticulum calcium levels and subsequently undergoes apoptosis in low serum conditions (Preston, G. A., Barrett, J. C., Biermann, J. A., and Murphy, E. (1997) Cancer Res. 57, 537–542). This decrease in endoplasmic reticulum calcium appears to be due, at least in part, to reduced capacitative calcium entry at the plasma membrane. Thus we investigated whether inhibition of capacitative calcium entry per se could reduce endoplasmic reticulum calcium and induce apoptosis of cells. We find that treatment with either SKF96365 (30–100 μm) or cell-impermeant 1,2-bis(o-amino-5-bromophenoxy)ethane-N,N,N′,N′-tetraacetic acid (5–10 mm) is able to induce apoptosis of cells in conditions where apoptosis does not normally occur. Because previous work has implicated vesicular trafficking as a mechanism of regulating capacitative calcium entry, we investigated whether disruption of vesicular trafficking could lead to decreased capacitative calcium entry and subsequent apoptosis of cells. Coincident with low serum-induced apoptosis, we observed an accumulation of vesicles within the cell, suggesting deregulated vesicle trafficking. Treatment of cells with bafilomycin (30–100 nm), an inhibitor of the endosomal proton ATPase, produced an accumulation of vesicles, decreased capacitative entry, and induced apoptosis. These data suggest that deregulation of vesicular transport results in reduced capacitative calcium entry which in turn results in apoptosis.

Essential role of Orai1 store-operated calcium channels in lactation

Significance All mammals, from platypuses to humans, produce relatively immature offspring that are wholly dependent on their mother’s milk for their postnatal growth and development. However, the dynamic signaling and molecular mechanisms responsible for the transport of key constituents (e.g., calcium) into milk and for alveolar unit contraction and milk ejection are not fully understood. Using genetically modified mouse models, we demonstrate that the store-operated Ca2+ channel Orai1 delivers over 50% of the calcium ions present in milk. We also reveal an unanticipated role of Orai1 as a master regulator of oxytocin-mediated alveolar unit contractility, milk ejection, and pup survival. These results provide a unique mechanistic insight into the fundamentally mammalian process of 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 Ca2+ channel subunit, Orai1, is required for both optimal Ca2+ 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 Ca2+ in mammary myoepithelial cells followed by slow, irregular Ca2+ oscillations. These oscillations, and not the initial Ca2+ 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 Ca2+ 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 Ca2+ 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).

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.

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

Lacrimal acinar cells from mice whose gene for Orai1 has been deleted have no detectable store‐operated Ca2+ entry, whether assessed by measurement of cytoplasmic Ca2+ changes or as a store‐operated current. Mice lacking Orai1 have diminished lacrimal fluid secretion in response to muscarinic–cholinergic stimulation. Mice lacking Orai1 also show diminished exocytosis, both in vivo and in vitro. The development and morphology of lacrimal glands, as well as responses not dependent on Ca2+ entry were unchanged in the knockout mice. The results demonstrate the central importance of store‐operated Ca2+ entry in lacrimal exocrine function, and suggest possible strategies for combating diseases associated with diminished lacrimal secretion.

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 Ca2+ 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 (STIM1fl/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.—Steinckwich, N., Myers, P., Janardhan, K. S., Flagler, N. D., King, D., Petranka, J. G., Putney, J. W. Role of the store‐operated calcium entry protein, STIM1, in neutrophil chemotaxis and infiltration into a murine model of psoriasis‐inflamed skin. FASEB J. 29, 3003‐3013 (2015). www.fasebj.org

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 CRAC channels, channels underlying store-operated Ca2+ entry (SOCE). However, the roles of these proteins in signaling and constructing other channels with biophysical properties distinct from CRAC channels are not known. We examined the hypothesis that STIM1 or Orai1 can interact with and regulate 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 signaling of TRPC channels. Specifically, Ca2+ entry seen after carbachol treatment in cells expressing TRPC1, 3, 5, or 6 were not enhanced by the co-expression of STIM1. Further, knockdown of STIM1 in cells expressing TRPC5 did not reduce TRPC5 activity, in contrast to published reports. Disruption of lipid rafts significantly attenuated TRPC3 activity, while having no effect on STIM1 localization or the development of ICRAC. This suggests that TRPC signaling and STIM1/Orai1 signaling occur in distinct plasma membrane domains. In vascular smooth muscle cells, arginine-vasopressin (AVP) activated non-selective cation currents, and single channel events recorded in cell-attached configuration from these cells detected a current with a slope conductance of 33.65 pS, similar to that published for TRPC6. Further, RT-PCR analysis of TRPC transcripts in A10 cells revealed the predominant expression of TRPC1 and TRPC6 mRNA. Using a membrane potential-sensitive dye as an assay, we determined that knockdown of either STIM1 or Orai1 had no effect on the function of this AVP-activated current, while store-operated entry was substantially reduced. Thus, both STIM1 and Orai1 appear to be specific molecular components of the ICRAC pathway and in our studies did not influence the function of exogenously or endogenously expressed TRPC channels.