**4.4. NF-κB, NFAT, CREB, and other transcription factors**

As we mentioned above, SOCE is essential for platelet activation, and it is known the key role of ORAI1 in response to thrombin [70, 71]. ORAI1 transcripts were found significantly reduced in platelets and megakaryocytes from SGK1-KO mice, and transfection of megakaryocyte with constitutively active SGK1 increased phosphorylation of the IκB kinase (IKKα/β), which phosphorylates the inhibitor protein IκBα, promoting nuclear translocation of NF-κB subunit p65 [59]. In addition, Eylenstein et al. defined, by chromatin immunoprecipitation (ChIP) experiments, the promoter regions accounting for NF-κB-sensitive genomic regulation of STIM1 and ORAI1 [72], supporting further the conclusion that upregulation of ORAI1 and STIM1 by SGK1-dependent NF-κB signaling leads to the upregulation of SOCE, which in turn upregulates expression of other transcription factors like fibroblast growth factor 23 (FGF23) [73]. Other transcription factors are known to regulate STIM1 expression, such as Wilms tumor suppressor 1 (WT1) and early growth response 1 (EGR1), which were found by analyzing the STIM1 promoter with the TESS search system (University of Pennsylvania) [74]. Finally, NEUROD2, a neurogenic transcription factor, has been described as a negatively regulator of STIM1 expression, an activity that limits the level of STIM1 in cortical neurons [75].

**4.3. Other kinases and pathways**

10 Calcium and Signal Transduction

It is known that Ca2+-influx is upregulated by phosphoinositide 3-kinase (PI3K) signaling in platelets [57, 58]. Because PI3K signaling involves activation of SGK1, and this kinase has been shown to be a stimulator of ORAI1 expression [59], it is accepted that PI3K modulates SOCE by upregulation of the CRAC channel. In B16B6 melanoma cells, constitutive activation of Src and PKB/Akt was revealed to be due to the activation of SOCE in lipid rafts, which promoted Ca2+-dependence of the Src activity to trigger tumor signaling events [60], as it reported for lung metastasis of melanoma cells in a xenograft mouse model [61]. However, this is not shared by other cancer cells, as in prostate PC3 cells, with low levels of active ERK1/2 due to constitutive activation of PKB/Akt, Src is fully activatable in a Ca2+-independent manner by epidermal growth factor (EGF) [19].

Protein kinase C (PKC) phosphorylates ORAI1 at residues Ser27 and Ser30 [62]. More precisely, Kawasaki et al. demonstrated that the knockdown of the isoform PKCβ led to an increase of Ca2+ influx, and that recombinant PKC phosphorylated ORAI1 in vitro and in vivo at these two amino acids, an effect that inhibited the Ca2+ transport through ORAI1 [62]. No other phospho-residues have been characterized in detail in ORAI1. In airway smooth muscle cells, rottlerin, a PKCδ-selective inhibitor, reduced phorbol esters-triggered SOCE, without affecting total levels of STIM1 and ORAI1 [63]. However, the mechanism of this inhibition remains to be elucidated. PKC also inhibited SOCE in hepatocytes treated with amiodarone, an experimental design to mimic the accumulation of lipids during steatosis [64]. Because selective inhibition of PKC reversed SOCE to normal values, it was concluded that lipid accumulation triggers PKC-dependent SOCE impairment. Also, accumulation of palmitate is cytotoxic in kidney cells, and high levels of palmitate triggered Ca2+ depletion in the ER, in addition to mitochondrial stress. This depletion is antagonized by the inhibition of fatty acid transporters, inhibition of phospholipase C (PLC), and inhibition of PKC [65]. Once again, the

mechanism that links PKC and the regulation of STIM1/ORAI1 remains elusive.

ing a positive loop to rise levels of mitochondrial fission.

**4.4. NF-κB, NFAT, CREB, and other transcription factors**

In 2011, Mungai et al. reported that hypoxia augmented cellular reactive oxygen species (ROS), without a significant alteration of energy charge values [66]. Hypoxia triggered an increase in [Ca2+]i, relocalization of STIM1 to ER-PM junctions, and phosphorylation of AMP-activated protein kinase (AMPK), in the absence of its upstream regulator LKB1 (liver kinase B1). These events were due to the ROS-dependent activation of ORAI1, which led to an increase in [Ca2+] i and activation of CaMKK2 (or CaMKKβ), an upstream activator of AMPK [66, 67]. Finally, a recent report from Yang et al. described how STIM1-ORAI1 mediated autophagy in endothelial progenitor cells exposed to oxidized low-density lipoprotein to mimic hypercholesterolemia. This treatment caused stimulation of Ca2+ influx mediated by STIM1-ORAI1, activation of CAMKK2 and decrease of mTOR activity with the subsequent activation of autophagy [68]. Similarly, in hepatocarcinoma cells, mitochondrial fission increased cytosolic Ca2+ levels that activated the NF-κB pathway, upregulating STIM1 expression and the subsequent SOCE [69]. The relative increase of [Ca2+]i also activated NFAT-dependent upregulation of Drp1, promot-

As we mentioned above, SOCE is essential for platelet activation, and it is known the key role of ORAI1 in response to thrombin [70, 71]. ORAI1 transcripts were found significantly reduced Regarding NF-κB, reciprocal regulation seems to link this transcription factor and SOCE. In addition to the activation of SOCE by NF-κB described above, Liu et al. reported that T-cell activation triggered by the binding of antigen to T-cell receptor stimulated SOCE and that this Ca2+ entry activated a PKCα-mediated phosphorylation of p65 NF-κB at Ser536, an event that controls nuclear localization and transcriptional activity of NF-κB [76].

STIM1 and ORAI1 are also well-known activators of the protein phosphatase calcineurin, which activates the nuclear factor of activated T cells (NFAT) [77]. Once NFAT becomes dephosphorylated by calcineurin the transcription factor is internalized into the nucleus. Indeed, the activation and nuclear translocation of NFAT was the reporter used by Feske et al. when they searched for regulators of SOCE using a Drosophila RNA interference screening, a report that led to the description of ORAI1 as the channel that mediates the Ca2+ releaseactivated Ca2+ current, or CRAC [78]. Because NFAT modulates the expression of a wide range of genes, it is involved in many pathways, and also in the regulation of the expression of other regulators of transcription, including IRF4, BATF, and Bcl-6 [79]. NFAT is not the only transcription factor activated by the axis STIM1-ORAI1-Ca2+/CaM-calcineurin because Ca2+ influx through ORAI1 stimulates the transcription factor EB (TFEB), promoting the activation of chemokines genes [80]. SOCE also activates the Ca2+/cAMP response element binding protein (CREB), a transcription factor that regulates expression of many genes, at least in cultured smooth muscle cells and intact arteries [81]. In this regard, it was observed that mitochondrial Ca2+ uptake was reduced in lymphocytes lacking STIM1 or ORAI1, an effect that was due to reduced mitochondrial Ca2+ uniporter (MCU) expression [82]. ChIP and promoter analyses revealed that CREB directly binds the MCU promoter, revealing that SOCE regulates the Ca2+ uptake capability of mitochondria by regulating Ca2+-dependent activation of CREB [82].

Ca2+ influx regulates myoblasts differentiation, and shortly after the molecular description of STIM and ORAI proteins, it was reported that silencing STIM1, Orai1, or Orai3 reduced SOCE and myoblast differentiation [83]. This positive effect on myoblasts correlated with the expression of MEF2 and myogenin, two transcription factors involved in skeletal muscle development, although it is still unclear the molecular pathway that links STIM1/ORAI1 and the activation of the transcription factors. In cerebellar granule neurons cultured in low concentration of extracellular potassium, mimicking resting conditions, SOCE promoted the degradation of transcription factor Sp4, a regulator of neuronal morphogenesis and function [84].

Another important molecular interactor of STIM1 is the hypoxia-inducible factor-1 alpha (HIF-1α), which is upregulated during hepatocarcinoma growth [85]. Li et al. found that HIF-1α directly controls STIM1 transcription, but also that STIM1-mediated SOCE is required for HIF-1α accumulation in hepatocarcinoma cells via activation of Ca2+/CaM-dependent protein kinase II, revealing a mutual dependence of STIM1 and HIF-1α in the regulation of Ca2+ transport and tumor growth [85].

**6. Future directions**

During the past decade, a significant progress was made regarding the molecular description of the proteins involved in store-operated Ca2+ entry. Although some details remain unclear,

Regulation of Calcium Signaling by STIM1 and ORAI1 http://dx.doi.org/10.5772/intechopen.78587 13

**Figure 2.** Activation of SOCE by STIM1/ORAI1 and pathways involved in SOCE-dependent signaling. Panel A: diverse stimuli that triggers the activation of the phospholipase C pathway, such as activation of EGF receptor (EGFR), stimulate the production of inositol 1,4,5-trisphosphate (IP3) which binds and activates IP3 receptor (IP3R) at the endoplasmic reticulum (ER). This activation leads to the release of Ca2+ from the ER, with the subsequent transient depletion of intraluminal [Ca2+] and the activation of STIM1. Ca2+-unbound STIM1 aggregates in oligomers and translocates to plasma membrane (PM)-ER junctions where it binds and activates ORAI1. Extracellular Ca2+ entry through ORAI1 activates multiple Ca2+-dependent targets, as shown in panel B, but also provides a Ca2+ source to replenish intraluminal Ca2+ levels. This replenishment is accomplished by the ER-Ca2+-ATPase which pumps Ca2+ into the ER lumen. Panel B: schematic illustration of the most important pathways regulated by STIM1/ORAI1. AC, adenylyl cyclase; GPCR, G

protein-coupled receptor; PM-STIM1, plasma membrane-resident STIM1; p-STIM1, phosphorylated STIM1.

High levels of ORAI1 and STIM1 are found in many types of cancer cells. In gastric cancer tumor progression, this higher expression is associated with a negative impact on survival rates of patients, an effect that was partially due to targeting expression of metastasis-associated in colon cancer-1 (MACC1) [86], an essential regulator of the transcription for the gene coding for the hepatocyte growth factor receptor, MET. Similarly, a recent report described that STIM1 promotes cell migration and the epithelial-to-mesenchymal transition (EMT) by activating TGF-β, Snail and Wnt/β-catenin pathways in prostate cancer cells [87].

Finally, an excellent report from Stephan Feske laboratory [54], described how SOCE is crucial for mitochondrial fatty acid oxidation, and that Ca2+ entry through ORAI1 was essential to activate adenylyl cyclase, cyclic AMP production, the transcriptional regulator peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) and peroxisome proliferator-activated receptor alpha (PPARα), which is mediated by the activation of CREB.
