The calcium channel Orai1 is required for osteoblast development: Studies in a chimeric mouse with variable in vivo Runx-cre deletion of Orai-1.

The calcium-selective ion channel Orai1 has a complex role in bone homeostasis, with defects in both bone production and resorption detected in Orai1 germline knock-out mice. To determine whether Orai1 has a direct, cell-intrinsic role in osteoblast differentiation and function, we bred Orai1 flox/flox (Orai1fl/fl) mice with Runx2-cre mice to eliminate its expression in osteoprogenitor cells. Interestingly, Orai1 was expressed in a mosaic pattern in Orai1fl/fl-Runx2-cre bone. Specifically, antibody labeling for Orai1 in vertebral sections was uniform in wild type animals, but patchy regions in Orai1fl/fl-Runx2-cre bone revealed Orai1 loss while in other areas expression persisted. Nevertheless, by micro-CT, bones from Orai1fl/fl-Runx2-cre mice showed reduced bone mass overall, with impaired bone formation identified by dynamic histomorphometry. Cortical surfaces of Orai1fl/fl-Runx2-cre vertebrae however exhibited patchy defects. In cell culture, Orai1-negative osteoblasts showed profound reductions in store-operated Ca2+ entry, exhibited greatly decreased alkaline phosphatase activity, and had markedly impaired substrate mineralization. We conclude that defective bone formation observed in the absence of Orai1 reflects an intrinsic role for Orai1 in differentiating osteoblasts.

Suppression of Ca2+ signaling enhances melanoma progression.

The role of store-operated Ca2+ entry (SOCE) in melanoma metastasis is highly controversial. To address this, we here examined UV-dependent metastasis, revealing a critical role for SOCE suppression in melanoma progression. UV-induced cholesterol biosynthesis was critical for UV-induced SOCE suppression and subsequent metastasis, although SOCE suppression alone was both necessary and sufficient for metastasis to occur. Further, SOCE suppression was responsible for UV-dependent differences in gene expression associated with both increased invasion and reduced glucose metabolism. Functional analyses further established that increased glucose uptake leads to a metabolic shift towards biosynthetic pathways critical for melanoma metastasis. Finally, examination of fresh surgically isolated human melanoma explants revealed cholesterol biosynthesis-dependent reduced SOCE. Invasiveness could be reversed with either cholesterol biosynthesis inhibitors or pharmacological SOCE potentiation. Collectively, we provide evidence that, contrary to current thinking, Ca2+ signals can block invasive behavior, and suppression of these signals promotes invasion and metastasis.

Mitochondrial pyruvate and fatty acid flux modulate MICU1-dependent control of MCU activity.

The tricarboxylic acid (TCA) cycle converts the end products of glycolysis and fatty acid ß-oxidation into the reducing equivalents NADH and FADH2 Although mitochondrial matrix uptake of Ca2+ enhances ATP production, it remains unclear whether deprivation of mitochondrial TCA substrates alters mitochondrial Ca2+ flux. We investigated the effect of TCA cycle substrates on MCU-mediated mitochondrial matrix uptake of Ca2+, mitochondrial bioenergetics, and autophagic flux. Inhibition of glycolysis, mitochondrial pyruvate transport, or mitochondrial fatty acid transport triggered expression of the MCU gatekeeper MICU1 but not the MCU core subunit. Knockdown of mitochondrial pyruvate carrier (MPC) isoforms or expression of the dominant negative mutant MPC1R97W resulted in increased MICU1 protein abundance and inhibition of MCU-mediated mitochondrial matrix uptake of Ca2+ We also found that genetic ablation of MPC1 in hepatocytes and mouse embryonic fibroblasts resulted in reduced resting matrix Ca2+, likely because of increased MICU1 expression, but resulted in changes in mitochondrial morphology. TCA cycle substrate-dependent MICU1 expression was mediated by the transcription factor early growth response 1 (EGR1). Blocking mitochondrial pyruvate or fatty acid flux was linked to increased autophagy marker abundance. These studies reveal a mechanism that controls the MCU-mediated Ca2+ flux machinery and that depends on TCA cycle substrate availability. This mechanism generates a metabolic homeostatic circuit that protects cells from bioenergetic crisis and mitochondrial Ca2+ overload during periods of nutrient stress.

Suppression of Ca2+ signals by EGR4 controls Th1 differentiation and anti-cancer immunity in vivo.

While the zinc finger transcription factors EGR1, EGR2, and EGR3 are recognized as critical for T-cell function, the role of EGR4 remains unstudied. Here, we show that EGR4 is rapidly upregulated upon TCR engagement, serving as a critical "brake" on T-cell activation. Hence, TCR engagement of EGR4-/- T cells leads to enhanced Ca2+ responses, driving sustained NFAT activation and hyperproliferation. This causes profound increases in IFNγ production under resting and diverse polarizing conditions that could be reversed by pharmacological attenuation of Ca2+ entry. Finally, an in vivo melanoma lung colonization assay reveals enhanced anti-tumor immunity in EGR4-/- mice, attributable to Th1 bias, Treg loss, and increased CTL generation in the tumor microenvironment. Overall, these observations reveal for the first time that EGR4 is a key regulator of T-cell differentiation and function.

The Ca2+ export pump PMCA clears near-membrane Ca2+ to facilitate store-operated Ca2+ entry and NFAT activation.

Ca2+ signals, which facilitate pluripotent changes in cell fate, reflect the balance between cation entry and export. We found that overexpression of either isoform of the Ca2+-extruding plasma membrane calcium ATPase 4 (PMCA4) pump in Jurkat T cells unexpectedly increased activation of the Ca2+-dependent transcription factor nuclear factor of activated T cells (NFAT). Coexpression of the endoplasmic reticulum-resident Ca2+ sensor stromal interaction molecule 1 (STIM1) with the PMCA4b splice variant further enhanced NFAT activity; however, coexpression with PMCA4a depressed NFAT. No PMCA4 splice variant dependence in STIM1 association was observed, whereas partner of STIM1 (POST) preferentially associated with PMCA4b over PMCA4a, which enhanced, rather than inhibited, PMCA4 function. A comparison of global and near-membrane cytosolic Ca2+ abundances during store-operated Ca2+ entry revealed that PMCA4 markedly depressed near-membrane Ca2+ concentrations, particularly when PMCA4b was coexpressed with STIM1. PMCA4b closely associated with both POST and the store-operated Ca2+ channel Orai1. Furthermore, POST knockdown increased the near-membrane Ca2+ concentration, inhibiting the global cytosolic Ca2+ increase. These observations reveal an unexpected role for POST in coupling PMCA4 to Orai1 to promote Ca2+ entry during T cell activation through Ca2+ disinhibition.

EGR-mediated control of STIM expression and function.

Ca2+ is a ubiquitous, dynamic and pluripotent second messenger with highly context-dependent roles in complex cellular processes such as differentiation, proliferation, and cell death. These Ca2+ signals are generated by Ca2+-permeable channels located on the plasma membrane (PM) and endoplasmic reticulum (ER) and shaped by PM- and ER-localized pumps and transporters. Differences in the expression of these Ca2+ homeostasis proteins contribute to cell and context-dependent differences in the spatiotemporal organization of Ca2+ signals and, ultimately, cell fate. This review focuses on the Early Growth Response (EGR) family of zinc finger transcription factors and their role in the transcriptional regulation of Stromal Interaction Molecule (STIM1), a critical regulator of Ca2+ entry in both excitable and non-excitable cells.

Novel STIM1-dependent control of Ca2+ clearance regulates NFAT activity during T-cell activation.

Antigen presentation to the T-cell receptor leads to sustained cytosolic Ca2+ elevation, which is critical for T-cell activation. We previously showed that in activated T cells, Ca2+ clearance is inhibited by the endoplasmic reticulum Ca2+ sensor stromal interacting molecule 1 (STIM1) via association with the plasma membrane Ca2+/ATPase 4 (PMCA4) Ca2+ pump. Having further observed that expression of both proteins is increased in activated T cells, the current study focused on mechanisms regulating both up-regulation of STIM1 and PMCA4 and assessing how this up-regulation contributes to control of Ca2+ clearance. Using a STIM1 promoter luciferase vector, we found that the zinc finger transcription factors early growth response (EGR) 1 and EGR4, but not EGR2 or EGR3, drive luciferase activity. We further found that neither STIM1 nor PMCA4 is up-regulated when both EGR1 and EGR4 are knocked down using RNA interference. Further, under these conditions, activation-induced Ca2+ clearance inhibition was eliminated with little effect on Ca2+ entry. Finally, we found that nuclear factor of activated T-cell (NFAT) activity is profoundly attenuated if Ca2+ clearance is not inhibited by STIM1. These findings reveal a critical role for STIM1-mediated control of Ca2+ clearance in NFAT induction during T-cell activation.-Samakai, E., Hooper, R., Martin, K. A., Shmurak, M., Zhang, Y., Kappes, D. J., Tempera, I., Soboloff, J. Novel STIM1-dependent control of Ca2+ clearance regulates NFAT activity during T-cell activation.

The heterogeneity of store-operated calcium entry in melanoma.

Calcium is a key regulator of many physiological processes that are perturbed in cancer, such as migration, proliferation and apoptosis. The proteins STIM and Orai mediate store-operated calcium entry (SOCE), the main pathway for calcium entry in non-excitable cells. Changes in the expression and function of STIM and Orai have been found in a range of cancer types and thus implicated in disease progression. Here we discuss the role of STIM, Orai and the SOCE pathway in the progression of melanoma and explore how the heterogeneous nature of melanoma may explain the lack of consensus in the field regarding the role of SOCE in the progression of this disease.

Novel Protein Kinase C-Mediated Control of Orai1 Function in Invasive Melanoma.

The incidence of malignant melanoma, a cancer of the melanocyte cell lineage, has nearly doubled in the past 20 years. Wnt5A, akey driver of melanoma invasiveness, induces Ca2 signals. To understand how store-operated calcium entry (SOCE) contributes to Wnt5A-induced malignancy in melanoma models, we examined the expression and function of STIM1 and Orai1 in patient-derived malignant melanoma cells, previously characterized as either highly invasive (metastatic) or noninvasive. Using both fluorescence microscopy and electrophysiological approaches, we show that SOCE is greatly diminished in invasive melanoma compared to its level in noninvasive cell types. However, no loss of expression of any members of the STIM and Orai families was observed in invasive melanoma cells. Moreover, overexpressed wild-type STIM1 and Orai1 failed to restore SOCE in invasive melanoma cells, and we observed no defects in their localization before or after store depletion in any of the invasive celllines. Importantly, however, we determined that SOCE was restored by inhibition of protein kinase C, a known downstream target of Wnt5A. Furthermore, coexpression of STIM1 with an Orai1 mutant insensitive to protein kinase C-mediated phosphorylation fully restored SOCE in invasive melanoma. These findings reveal a level of control for STIM/Orai function in invasive melanoma not previously reported.

Photoaffinity labeling of nicotinic acid adenine dinucleotide phosphate (NAADP) targets in mammalian cells.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is an agonist-generated second messenger that releases Ca(2+) from intracellular acidic Ca(2+) stores. Recent evidence has identified the two-pore channels (TPCs) within the endolysosomal system as NAADP-regulated Ca(2+) channels that release organellar Ca(2+) in response to NAADP. However, little is known about the mechanism coupling NAADP binding to calcium release. To identify the NAADP binding site, we employed a photoaffinity labeling method using a radioactive photoprobe based on 5-azido-NAADP ([(32)P-5N(3)]NAADP) that exhibits high affinity binding to NAADP receptors. In several systems that are widely used for studying NAADP-evoked Ca(2+) signaling, including sea urchin eggs, human cell lines (HEK293, SKBR3), and mouse pancreas, 5N(3)-NAADP selectively labeled low molecular weight sites that exhibited the diagnostic pharmacology of NAADP-sensitive Ca(2+) release. Surprisingly, we were unable to demonstrate labeling of endogenous, or overexpressed, TPCs. Furthermore, labeling of high affinity NAADP binding sites was preserved in pancreatic samples from TPC1 and TPC2 knock-out mice. These photolabeling data suggest that an accessory component within a larger TPC complex is responsible for binding NAADP that is unique from the core channel itself. This observation necessitates critical evaluation of current models of NAADP-triggered activation of the TPC family.

Membrane topology of NAADP-sensitive two-pore channels and their regulation by N-linked glycosylation.

Two-pore channels (TPCs) localize to the endolysosomal system and have recently emerged as targets for the Ca(2+)-mobilizing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). However, their membrane topology is unknown. Using fluorescence protease protection assays, we show that human TPC1 and TPC2 possess cytosolic N and C termini and therefore an even number of transmembrane regions. Fluorophores placed at position 225 or 347 in TPC1, or 339 in TPC2 were also cytosolic, whereas a fluorophore at position 628 in TPC1 was luminal. These data together with sequence similarity to voltage-gated Ca(2+) and Na(+) channels, and unbiased in silico predictions are consistent with a topology in which two homologous domains are present, each comprising 6 transmembrane regions and a re-entrant pore loop. Immunocytochemical analysis of selectively permeabilized cells using antipeptide antibodies confirmed that the C-terminal tails of recombinant TPCs are cytosolic and that residues 240-254 of TPC2 prior to putative pore 1 are luminal. Both TPC1 and TPC2 are N-glycosylated with residues 599, 611, and 616 contributing to glycosylation of TPC1. This confirms the luminal position of these residues, which immediately precede the putative pore loop of the second domain. Mutation of all three glycosylation sites in TPC1 enhances NAADP-evoked cytosolic Ca(2+) signals. Our data establish essential features of the topology of two-pore channels.

An NAADP-gated two-pore channel targeted to the plasma membrane uncouples triggering from amplifying Ca2+ signals.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a ubiquitous messenger proposed to stimulate Ca(2+) release from acidic organelles via two-pore channels (TPCs). It has been difficult to resolve this trigger event from its amplification via endoplasmic reticulum Ca(2+) stores, fuelling speculation that archetypal intracellular Ca(2+) channels are the primary targets of NAADP. Here, we redirect TPC2 from lysosomes to the plasma membrane and show that NAADP evokes Ca(2+) influx independent of ryanodine receptors and that it activates a Ca(2+)-permeable channel whose conductance is reduced by mutation of a residue within a putative pore. We therefore uncouple TPC2 from amplification pathways and prove that it is a pore-forming subunit of an NAADP-gated Ca(2+) channel.

The strength of suture configurations in arthroscopic rotator cuff repair.

PURPOSE: This study evaluated the strength of different suture configurations with the use of a recently introduced arthroscopic suture passer (ExpressSew; Surgical Solutions, Valencia, CA). METHODS: The ultimate tensile strength and mode of failure of each suture configuration were repeatedly tested on a validated porcine rotator cuff tendon model, with the use of standard suture material (No. 2 FiberWire [Arthrex, Naples, FL]) passed with ExpressSew and tested on a Hounsfield digital tensiometer type H20K-W (Tinius Olsen, Hersham, PA). RESULTS: The strongest construct was 2 mattress sutures (mean, 169 N; standard deviation, 56.1 N); this was followed in strength by a single modified Kessler suture (mean, 161 N; standard deviation, 16.9 N), 4 simple sutures (mean, 155 N; standard deviation, 27.3 N), and finally, a single Mason-Allen suture (mean, 140 N; standard deviation, 28.6 N). CONCLUSIONS: Study results show little difference in strength for varying complexities of suture configurations. In simple terms, no demonstrable difference was seen in the strength of construct, whether the surgeon used 4 simple sutures, 2 mattress sutures, or 1 grasping suture. CLINICAL RELEVANCE: This study allows the surgeon to justify use of the simplest configuration of suture passage that works in his hands, for the purpose of attaining a reliable and repeatable repair of the rotator cuff through arthroscopic methods.