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.

The function of the calcium channel Orai1 in osteoclast development.

To determine the intrinsic role of Orai1 in osteoclast development, Orai1-floxed mice were bred with LysMcre mice to delete Orai1 from the myeloid lineage. PCR, in situ labelling and Western analysis showed Orai1 deletion in myeloid-lineage cells, including osteoclasts, as expected. Surprisingly, bone resorption was maintained in vivo, despite loss of multinucleated osteoclasts; instead, a large number of mononuclear cells bearing tartrate resistant acid phosphatase were observed on cell surfaces. An in vitro resorption assay confirmed that RANKL-treated Orai1 null cells, also TRAP-positive but mononuclear, degraded matrix, albeit at a reduced rate compared to wild type osteoclasts. This shows that mononuclear osteoclasts can degrade bone, albeit less efficiently. Further unexpected findings included that Orai1fl/fl -LysMcre vertebrae showed slightly reduced bone density in 16-week-old mice, despite Orai1 deletion only in myeloid cells; however, this mild difference resolved with age. In summary, in vitro analysis showed a severe defect in osteoclast multinucleation in Orai1 negative mononuclear cells, consistent with prior studies using less targeted strategies, but with evidence of resorption in vivo and unexpected secondary effects on bone formation leaving bone mass largely unaffected.

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.

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 critical role of STIM1-dependent Ca2+ signalling during T-cell development and activation.

T lymphocytes are key cellular effectors of adaptive immunity able to recognize a virtually limitless number of antigenic peptides and mount an immune response. Ca(2+) signals are crucial to the development and activation of T cells and Stromal Interaction Molecule 1 (STIM1) has been identified as a critical modulator of intracellular Ca(2+) levels in T cells. Although the role of STIM1 in T cell activation has been extensively investigated, the role of STIM1 in T cell development has been somewhat controversial. Indeed, deficiencies in STIM1 expression and function lead to both developmental defects associated with the development of autoimmunity yet also interfere with T cell activation leading to severe combined immunodeficiency signifying a multifaceted role of STIM1 in T cell physiology and pathophysiology.

Multifaceted roles of STIM proteins.

Stromal interaction molecules (STIM1 and STIM2) are critical components of store-operated calcium entry. Sensing depletion of endoplasmic reticulum (ER) Ca(2+) stores, STIM couples with plasma membrane Orai channels, resulting in the influx of Ca(2+) across the PM into the cytosol. Although best recognized for their primary role as ER Ca(2+) sensors, increasing evidence suggests that STIM proteins have a broader variety of sensory capabilities than first envisaged, reacting to cell stressors such as oxidative stress, temperature, and hypoxia. Further, the array of partners for STIM proteins is now understood to range far beyond the Orai channel family. Here we discuss the implications of STIM's expanding role, both as a stress sensor and a general modulator of multiple physiological processes in the cell.

STIM1 is required for attenuation of PMCA-mediated Ca2+ clearance during T-cell activation.

T-cell activation involves a complex signalling cascade uniquely dependent on elevated cytosolic Ca(2+) levels. Further, the spatiotemporal characteristics of this Ca(2+) signal play a critical role in this process via selective activation of transcription factors. In T cells, store-operated Ca(2+) entry (SOCe) is the primary Ca(2+) influx pathway; however, cytosolic Ca(2+) concentration depends upon the balance between Ca(2+) influx and extrusion. The plasma membrane Ca(2+) ATPase (PMCA) has previously been identified as a critical player in Ca(2+) clearance in T cells. Here, we provide data revealing both functional and physical links between the activation of stromal interacting molecule 1 (STIM1) and PMCA-mediated Ca(2+) clearance. Due to the ubiquitous expression of both STIM1 and PMCA, these findings have wide-ranging implications for Ca(2+) signalling in multiple cell types.

Structure and function of multiple Ca2+-binding sites in a K+ channel regulator of K+ conductance (RCK) domain.

Regulator of K(+) conductance (RCK) domains control the activity of a variety of K(+) transporters and channels, including the human large conductance Ca(2+)-activated K(+) channel that is important for blood pressure regulation and control of neuronal firing, and MthK, a prokaryotic Ca(2+)-gated K(+) channel that has yielded structural insight toward mechanisms of RCK domain-controlled channel gating. In MthK, a gating ring of eight RCK domains regulates channel activation by Ca(2+). Here, using electrophysiology and X-ray crystallography, we show that each RCK domain contributes to three different regulatory Ca(2+)-binding sites, two of which are located at the interfaces between adjacent RCK domains. The additional Ca(2+)-binding sites, resulting in a stoichiometry of 24 Ca(2+) ions per channel, is consistent with the steep relation between [Ca(2+)] and MthK channel activity. Comparison of Ca(2+)-bound and unliganded RCK domains suggests a physical mechanism for Ca(2+)-dependent conformational changes that underlie gating in this class of channels.