The process of store-operated Ca2+ entry (SOCE), whereby Ca2+ influx across the plasma membrane is activated in response to depletion of intracellular Ca2+ stores in the endoplasmic reticulum (ER), has been under investigation for greater than 25 years; however, only in the past 5 years have we come to understand this mechanism at the molecular level. A surge of recent experimentation indicates that STIM molecules function as Ca2+ sensors within the ER that, upon Ca2+ store depletion, rearrange to sites very near to the plasma membrane. At these plasma membrane-ER junctions, STIM interacts with and activates SOCE channels of the Orai family. The molecular and biophysical data that have led to these findings are discussed in this review, as are several controversies within this rapidly expanding field.

Summary When cells are activated by calcium-mobilizing agonists at low, physiological concentrations, the resulting calcium signals generally take the form of repetitive regenerative discharges of stored calcium, termed calcium oscillations [1]. These intracellular calcium oscillations have long fascinated biologists as representing a mode of digitized intracellular signaling. Recent work has highlighted the role of calcium influx as an essential component of calcium oscillations [2]. This influx occurs through a process known as store-operated calcium entry which is initiated by calcium sensor proteins in the endoplasmic reticulum, STIM1 and STIM2 [3]. STIM2 is activated by changes in endoplasmic reticulum calcium near the resting level, while a threshold of calcium depletion is required to activate STIM1 [4]. In this study, we show that, surprisingly, it is STIM1 and not STIM2 that is exclusively involved in calcium entry during calcium oscillations. The implication is that each oscillation produces a transient drop in endoplasmic reticulum calcium that is sufficient to transiently activate STIM1. This transient activation of STIM1 can be observed in some cells by total internal reflection fluorescence microscopy. This arrangement nicely provides a clearly defined and unambiguous signaling system, translating a digital calcium release signal into calcium influx that can signal to downstream effectors.

It has long been known that many bone diseases, including osteoporosis, involve abnormalities in osteoclastic bone resorption. As a result, there has been intense study of the mechanisms that regulate both the differentiation and bone resorbing function of osteoclast cells. Calcium (Ca2+) signaling appears to play a critical role in the differentiation and functions of osteoclasts. Cytoplasmic Ca2+ oscillations occur during RANKL-mediated osteoclastogenesis. Ca2+ oscillations provide a digital Ca2+ signal that induces osteoclasts to up-regulate and autoamplify nuclear factor of activated T cells c1 (NFATc1), a Ca2+/calcineurin-dependent master regulator of osteoclastogenesis. Here we review previous studies on Ca2+ signaling in osteoclasts as well as recent breakthroughs in understanding the basis of RANKL-induced Ca2+ oscillations, and we discuss possible molecular players in this specialized Ca2+ response that appears pivotal for normal bone function.

The clustering of signaling molecules at specialized cellular sites allows cells to effectively convert extracellular signals into intracellular signals and to produce a concerted functional output with specific temporal and spatial patterns. A prime example for these molecules and their effects on cellular signaling are the postsynaptic density proteins of the central nervous system. Recently, one group of these proteins, the Vesl/Homer protein family has received increased attention because of its unique molecular properties that allow both the clustering and functional modulation of a plethora of different binding proteins. Within multiprotein signaling complexes, Vesl/Homer proteins influence proteins as diverse as metabotropic glutamate receptors; transient receptor potential channels; intra-cellular calcium channels; the scaffolding protein, Shank; small GTPases; transcription factors; and cytoskeletal proteins. Furthermore, interaction with such functionally relevant proteins also links Vesl/Homer proteins indirectly to an even larger group of cellular effector proteins, putting the Vesl/Homer proteins at the crossroads of several critical intracellular signaling processes. In addition to the initial reports of Vesl/Homer protein expression in the central nervous system, members of this protein family have now been identified in other excitable cells in various muscle types and in a large number of nonexcitable cells. The widespread expression of Vesl/Homer proteins in different organs and their functional importance in cellular protein signaling complexes is further evidenced by their conservation in organisms from Drosophila to humans.

Bone diseases such as postmenopausal osteoporosis are primarily caused by excessive formation and activity of osteoclasts (OCLs). Receptor activator of nuclear factor-κB ligand (RANKL) is a key initiating cytokine for OCL differentiation and function. RANKL induces calcium (Ca(2+)) oscillations, resulting in selective and robust induction of nuclear factor of activated T cells c1 (NFATc1), a Ca(2+)-responsive transcription factor that drives osteoclastogenesis. Store-operated Ca(2+) entry (SOCE) is a major Ca(2+) influx pathway in most nonexcitable cell types and is activated by any stimulus that depletes Ca(2+) stores in the endoplasmic reticulum. Although the role of Orai1, a SOCE channel in the plasma membrane, in maintaining Ca(2+) oscillations and transactivation of NFAT in other cell types is well known, its contribution to osteoclastogenesis remains unclear. We show here that silencing of the Orai1 gene with viral delivery of shRNA reduces SOCE and inhibits RANKL-induced osteoclastogenesis of RAW264.7 cells, a murine monocyte/macrophage cell line, by suppressing the induction of NFATc1. This was accompanied by defective induction of OCL-specific genes, such as tartrate-resistant acid phosphatase and immunoreceptor OCL-associated receptor, which are known to be direct transcriptional targets of NFATc1 during osteoclastogenesis. In addition, maturation of OCLs was abrogated by defective cell fusion of pre-OCLs depleted of Orai1, consistent with defective RANKL-mediated induction of d2 isoform of vacuolar ATPase V(o) domain that is involved in cell fusion of pre-OCLs. We found that the functional bone resorbing capacity was severely impaired in OCLs depleted of Orai1, potentially related to the observed decrease in the induction of cathepsin K, a major bone matrix degrading protease. Our results indicate that Orai1 plays a critical role in the differentiation and function of OCLs, suggesting that Orai1 might be a potential therapeutic target for the treatment or prevention of bone loss caused by OCLs.

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The fusion of monocyte/macrophage lineage cells into fully active, multinucleated, bone resorbing osteoclasts is a complex cell biological phenomenon that utilizes specialized proteins. OC-STAMP, a multi-pass transmembrane protein, has been shown to be required for pre-osteoclast fusion and for optimal bone resorption activity. A previously reported knockout mouse model had only mononuclear osteoclasts with markedly reduced resorption activity in vitro, but with paradoxically normal skeletal micro-CT parameters. To further explore this and related questions, we used mouse ES cells carrying a gene trap allele to generate a second OC-STAMP null mouse strain. Bone histology showed overall normal bone form with large numbers of TRAP-positive, mononuclear osteoclasts. Micro-CT parameters were not significantly different between knockout and wild type mice at 2 or 6 weeks old. At 6 weeks, metaphyseal TRAP-positive areas were lower and mean size of the areas were smaller in knockout femora, but bone turnover markers in serum were normal. Bone marrow mononuclear cells became TRAP-positive when cultured with CSF-1 and RANKL, but they did not fuse. Expression levels of other osteoclast markers, such as cathepsin K, carbonic anhydrase II, and NFATc1, were not significantly different compared to wild type. Actin rings were present, but small, and pit assays showed a 3.5-fold decrease in area resorbed. Restoring OC-STAMP in knockout cells by lentiviral transduction rescued fusion and resorption. N- and C-termini of OC-STAMP were intracellular, and a predicted glycosylation site was shown to be utilized and to lie on an extracellular loop. The site is conserved in all terrestrial vertebrates and appears to be required for protein stability, but not for fusion. Based on this and other results, we present a topological model of OC-STAMP as a 6-transmembrane domain protein. We also contrast the osteoclast-specific roles of OC- and DC-STAMP with more generalized cell fusion mechanisms.