Targeting CAMKK2 and SOC Channels as a Novel Therapeutic Approach for Sensitizing Acute Promyelocytic Leukemia Cells to All-Trans Retinoic Acid

Calcium ions (Ca2+) play important and diverse roles in the regulation of autophagy, cell death and differentiation. Here, we investigated the impact of Ca2+ in regulating acute promyelocytic leukemia (APL) cell fate in response to the anti-cancer agent all-trans retinoic acid (ATRA). We observed that ATRA promotes calcium entry through store-operated calcium (SOC) channels into acute promyelocytic leukemia (APL) cells. This response is associated with changes in the expression profiles of ORAI1 and STIM1, two proteins involved in SOC channels activation, as well as with a significant upregulation of several key proteins associated to calcium signaling. Moreover, ATRA treatment of APL cells led to a significant activation of calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) and its downstream effector AMP-activated protein kinase (AMPK), linking Ca2+ signaling to autophagy. Pharmacological inhibition of SOC channels and CAMKK2 enhanced ATRA-induced cell differentiation and death. Altogether, our results unravel an ATRA-elicited signaling pathway that involves SOC channels/CAMKK2 activation, induction of autophagy, inhibition of cellular differentiation and suppression of cell death. We suggest that SOC channels and CAMKK2 may constitute novel drug targets for potentiating the anti-cancer effect of ATRA in APL patients.

Blockage of Store-Operated Ca2+ Influx by Synta66 is Mediated by Direct Inhibition of the Ca2+ Selective Orai1 Pore

The Ca2+ sensor STIM1 and the Ca2+ channel Orai1 that form the store-operated Ca2+ (SOC) channel complex are key targets for drug development. Selective SOC inhibitors are currently undergoing clinical evaluation for the treatment of auto-immune and inflammatory responses and are also deemed promising anti-neoplastic agents since SOC channels are linked with enhanced cancer cell progression. Here, we describe an investigation of the site of binding of the selective inhibitor Synta66 to the SOC channel Orai1 using docking and molecular dynamics simulations, and live cell recordings. Synta66 binding was localized to the extracellular site close to the transmembrane (TM)1 and TM3 helices and the extracellular loop segments, which, importantly, are adjacent to the Orai1-selectivity filter. Synta66-sensitivity of the Orai1 pore was, in fact, diminished by both Orai1 mutations affecting Ca2+ selectivity and permeation of Na+ in the absence of Ca2+. Synta66 also efficiently blocked SOC in three glioblastoma cell lines but failed to interfere with cell viability, division and migration. These experiments provide new structural and functional insights into selective drug inhibition of the Orai1 Ca2+ channel by a high-affinity pore blocker.

TRPC Channels in the SOCE Scenario

Transient receptor potential (TRP) proteins form non-selective Ca2+ permeable channels that contribute to the modulation of a number of physiological functions in a variety of cell types. Since the identification of TRP proteins in Drosophila, it is well known that these channels are activated by stimuli that induce PIP2 hydrolysis. The canonical TRP (TRPC) channels have long been suggested to be constituents of the store-operated Ca2+ (SOC) channels; however, none of the TRPC channels generate Ca2+ currents that resemble ICRAC. STIM1 and Orai1 have been identified as the components of the Ca2+ release-activated Ca2+ (CRAC) channels and there is a body of evidence supporting that STIM1 is able to gate Orai1 and TRPC1 in order to mediate non-selective cation currents named ISOC. STIM1 has been found to interact to and activate Orai1 and TRPC1 by different mechanisms and the involvement of TRPC1 in store-operated Ca2+ entry requires both STIM1 and Orai1. In addition to the participation of TRPC1 in the ISOC currents, TRPC1 and other TRPC proteins might play a relevant role modulating Orai1 channel function. This review summarizes the functional role of TRPC channels in the STIM1–Orai1 scenario.

Spongionella Secondary Metabolites Regulate Store Operated Calcium Entry Modulating Mitochondrial Functioning in SH-SY5Y Neuroblastoma Cells

Background/Aims: The effect of four secondary metabolites isolated from sponge Spongionella, gracilins H, A, L and tetrahydroaplysulphurin-1 on Calcium ion (Ca2+) fluxes were studied in SH-SY5Y neuroblastoma cells. Methods and Results: These compounds did not modify cytosolic baseline Ca2+-levels. Nevertheless, when cytosolic Ca2+-influx through store operated calcium channels (SOC channels) was stimulated with Thapsigargin (Tg), a strong inhibition was observed in the presence of gracilin A, gracilin L and tetrahydroaplysulphurin-1. Since these compounds were able to protect mitochondria from oxidative stress, the role of this organelle in the Ca2+-influx inhibition was tested. In this sense, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) and Cyclosporine A (CsA) were used. Surprisingly, both the inhibitory effect over Tg-sensitive stores and Ca2+ influx through SOC channels produced by FCCP were abolished with different potencies by Spongionella compounds in a similar way than CsA. CsA is able to avoid Mitochondrial Permeability Transition Pore (mPTP) opening. As well as CsA, Spongionella compounds reverted mPTP opening induced by FCCP. In the case of CsA the mPTP blockade is due to the direct binding to Cyclophilin D (Cyp D), a mitochondrial matrix protein. This association was also observed between gracilin L and tetrahydroaplysulphurin-1 and Cyp D. Therefore, Spongionella compounds modulate mitochondrial activity by preventing mPTP opening by binding to Cyp D. Conclusions: These effects make Spongionella compounds as new family of compounds with promising activity in human diseases where mitochondrial alterations are implicated.

Calcium-dependent and calcium-sensitizing pathways in the mature and immature ductus arteriosus

Studies performed in sheep and baboons have shown that after birth, the normoxic muscle media of ductus arteriosus (DA) becomes profoundly hypoxic as it constricts and undergoes anatomic remodeling. We used isolated fetal lamb DA (pretreated with inhibitors of prostaglandin and nitric oxide production) to determine why the immature DA fails to remain tightly constricted during the hypoxic phase of remodeling. Under normoxic conditions, mature DA constricts to 70% of its maximal active tension (MAT). Half of its normoxic tension is due to Ca2+ entry through calcium L-channels and store-operated calcium (SOC) channels. The other half is independent of extracellular Ca2+ and is unaffected by inhibitors of sarcoplasmic reticulum (SR) Ca2+ release (ryanodine) or reuptake [cyclopiazonic acid (CPA)]. The mature DA relaxes slightly during hypoxia (to 60% MAT) due to decreases in calcium L-channel-mediated Ca2+ entry. Inhibitors of Rho kinase and tyrosine kinase inhibit both Ca2+-dependent and Ca2+-independent DA tension. Although Rho kinase activity may increase during gestation, immature DA develop lower tensions than mature DA, primarily because of differences in the way they process Ca2+. Calcium L-channel expression increases with advancing gestation. Under normoxic conditions, differences in calcium L-channel-mediated Ca2+ entry account for differences in tension between immature (60% MAT) and mature (70% MAT) DA. Under hypoxic conditions, differences in both calcium L-channel-dependent and calcium L-channel-independent Ca2+ entry, account for differences in tension between immature (33% MAT) and mature (60% MAT) DA. Stimulation of Ca2+ entry through reverse-mode Na+/Ca2+ exchange or CPA-induced SOC channel activity constrict the DA and eliminate differences between immature and mature DA during both hypoxia and normoxia.

Function of a STIM1 Homologue in C. elegans: Evidence that Store-operated Ca2+ Entry Is Not Essential for Oscillatory Ca2+ Signaling and ER Ca2+ Homeostasis

1,4,5-trisphosphate (IP3)-dependent Ca2+ signaling regulates gonad function, fertility, and rhythmic posterior body wall muscle contraction (pBoc) required for defecation in Caenorhabditis elegans. Store-operated Ca2+ entry (SOCE) is activated during endoplasmic reticulum (ER) Ca2+ store depletion and is believed to be an essential and ubiquitous component of Ca2+ signaling pathways. SOCE is thought to function to refill Ca2+ stores and modulate Ca2+ signals. Recently, stromal interaction molecule 1 (STIM1) was identified as a putative ER Ca2+ sensor that regulates SOCE. We cloned a full-length C. elegans stim-1 cDNA that encodes a 530–amino acid protein with ∼21% sequence identity to human STIM1. Green fluorescent protein (GFP)–tagged STIM-1 is expressed in the intestine, gonad sheath cells, and spermatheca. Knockdown of stim-1 expression by RNA interference (RNAi) causes sterility due to loss of sheath cell and spermatheca contractile activity required for ovulation. Transgenic worms expressing a STIM-1 EF-hand mutant that constitutively activates SOCE in Drosophila and mammalian cells are sterile and exhibit severe pBoc arrhythmia. stim-1 RNAi dramatically reduces STIM-1∷GFP expression, suppresses the EF-hand mutation–induced pBoc arrhythmia, and inhibits intestinal store-operated Ca2+ (SOC) channels. However, stim-1 RNAi surprisingly has no effect on pBoc rhythm, which is controlled by intestinal oscillatory Ca2+ signaling, in wild type and IP3 signaling mutant worms, and has no effect on intestinal Ca2+ oscillations and waves. Depletion of intestinal Ca2+ stores by RNAi knockdown of the ER Ca2+ pump triggers the ER unfolded protein response (UPR). In contrast, stim-1 RNAi fails to induce the UPR. Our studies provide the first detailed characterization of STIM-1 function in an intact animal and suggest that SOCE is not essential for certain oscillatory Ca2+ signaling processes and for maintenance of store Ca2+ levels in C. elegans. These findings raise interesting and important questions regarding the function of SOCE and SOC channels under normal and pathophysiological conditions.

Calcium Signaling Mediated by P2Y Receptors in Mouse Taste Cells

Evidence implicates a number of neuroactive substances and their receptors in mediating complex cell-to-cell communications in the taste bud. Recently, we found that ATP, a ubiquitous neurotransmitter/neuromodulator, mobilizes intracellular Ca2+ in taste cells by activating P2Y receptors. Here, P2Y receptor-cellular response coupling was characterized in detail using single cell ratio photometry and the inhibitory analysis. The sequence of underlying events was shown to include ATP-dependent activation of PLC, IP3 production, and IP3 receptor-mediated Ca2+ release followed by Ca2+ influx. Data obtained favor SOC channels rather than receptor-operated channels as a pathway for Ca2+ influx that accompanies Ca2+ release. Intracellular Ca2+ mobilized by ATP is apparently extruded by the plasma membrane Ca2+-ATPase, while a contribution of the Na+/Ca2+ exchange and other mechanisms of Ca2+ clearance is negligible. Cyclic AMP-dependent phosphorylation is likely to control a gain of the phosphoinositide cascade involved in ATP transduction. ATP-responsive taste cells are abundant in circumvallate, foliate, and fungiform papillae. Taken together, our observations point to a putative role for ATP as a neurotransmitter operative in the taste bud.