Intracellular Ca can compensate for the lack of NADPH oxidase-derived ROS in endothelial cells

The aim of the present study is to determine the role of intracellular Ca²⁺ in VEGF signaling. We demonstrate that reduction in Ca²⁺ by chelating compound BAPTA-AM or by IP₃-endoplasmic reticulum blocker 2-APB selectively inhibited VEGF-induced activation of c-Src-PI3K-Akt but not ERK1/2 in human coronary artery endothelial cells (HCAEC). We also show that the selective inhibitory effects of NADPH oxidase knockdown on VEGF-mediated activation of c-Src-PI3K-Akt signaling and cell proliferation in HCAEC can be reversed by increase in intracellular Ca²⁺. These results suggest an essential role for Ca²⁺ in redox-dependent selective activation of c-Src-PI3K-Akt and endothelial cell proliferation.     

Role of SOCE architects STIM and Orai proteins in Cell Death

Calcium (Ca²⁺) signaling plays a critical role in regulating plethora of cellular functions including cell survival, proliferation and migration. The perturbations in cellular Ca²⁺ homeostasis can lead to cell death either by activating autophagic pathways or through induction of apoptosis. Endoplasmic reticulum (ER) is the major storehouse of Ca²⁺ within cells and a number of physiological agonists mediate ER Ca²⁺ release by activating IP₃ receptors (IP₃R). This decrease in ER Ca²⁺ levels is sensed by STIM, which physically interacts and activates plasma membrane Ca²⁺ selective Orai channels. Emerging literature implicates a key role for STIM1, STIM2, Orai1 and Orai3 in regulating both cell survival and death pathways. In this review, we will retrospect the work highlighting the role of STIM and Orai homologs in regulating cell death signaling. We will further discuss the rationales that could explain the dual role of STIM and Orai proteins in regulating cell fate decisions.     

The role of Ca2+ mobilization and heterotrimeric G protein activation in mediating tyrosine phosphorylation signaling patterns in vascular smooth muscle cells

This work investigated the role of Ca²⁺ mobilization and heterotrimeric G protein activation in mediating angiotensin II-dependent tyrosine phosphorylation signaling patterns. We demonstrate that the predominant, angiotensin II-dependent, tyrosine phosphorylation signaling patterns seen in vascular smooth muscle cells are blocked by the intracellular Ca²⁺ chelator BAPTA-AM, but not by the Ca²⁺ channel blocker verapamil. Activation of heterotrimeric G proteins with NaF resulted in a divergent signaling effect; NaF treatment was sufficient to increase tyrosine phosphorylation levels of some proteins independent of angiotensin II treatment. In the same cells, NaF alone had no effect on other cellular proteins, but greatly potentiated the ability of angiotensin II to increase the tyrosine phosphorylation levels of these proteins. Two proteins identified in these studies were paxillin and Jak2. We found that NaF treatment alone, independent of angiotensin II stimulation, was sufficient to increase the tyrosine phosphorylation levels of paxillin. Furthermore, the ability of either NaF and/or angiotensin II to increase tyrosine phosphorylation levels of paxillin is critically dependent on intracellular Ca²⁺. In contrast, angiotensin II-mediated Jak2 tyrosine phosphorylation was independent of intracellular Ca²⁺ mobilization and extracellular Ca²⁺ entry. Thus, our data suggest that angiotensin II-dependent tyrosine phosphorylation signaling cascades are mediated through a diverse set of signaling pathways that are partially dependent on Ca²⁺ mobilization and heterotrimeric G protein activation.     

Calcium is essential for fructan synthesis induction mediated by sucrose in wheat

The role of Ca²⁺ in the induction of enzymes involved in fructan synthesis (FSS) mediated by sucrose was studied in wheat (Triticum aestivum). Increase of FSS enzyme activity and induction of the expression of their coding genes by sucrose were inhibited in leaf blades treated with chelating agents (EDTA, EGTA and BAPTA). Ca²⁺ channel blockers (lanthanum chloride and ruthenium red) also inhibited the FSS response to sucrose, suggesting the participation of Ca²⁺ from both extra- and intra- cellular stores. Sucrose induced a rapid Ca²⁺ influx into the cytosol in wheat leaf and root tissues, shown with the Ca²⁺ sensitive fluorescent probe Fluo-3/AM ester. Our results support the hypothesis that calcium is a component of the sucrose signaling pathway that leads to the induction of fructan synthesis.     

Ca(2+)-dependent in vivo protein phosphorylation and encystment induction in the ciliated protozoan Colpoda cucullus

Encystment induction of Colpoda cucullus is promoted by an increase in external Ca²⁺ and overpopulation of Colpoda vegetative cells. Using phos-tag detection assays, the present study revealed that the in vivo phosphorylation level in several proteins [33 kDa, 37 kDa, 37.5 kDa, 43 kDa, 47 kDa, 49 kDa, etc.] was raised when the vegetative cells were stimulated by overpopulation to encyst in a medium containing 0.1 mM Ca²⁺ or without the addition of Ca²⁺. Both overpopulation-mediated encystment induction and protein phosphorylation were suppressed by the addition of EGTA. Ca²⁺/overpopulation-stimulated encystment induction and protein phosphorylation were also suppressed by the addition of BAPTA-AM. These results suggest that the Ca²⁺ inflow promoted by cell-to-cell stimulation due to overpopulation may activate signaling pathways involving protein phosphorylation and encystment induction. In the presence of cAMP-AM, the phosphorylation levels of 33 kDa, 37 kDa, 37.5 kDa, 43 kDa, 47 kDa and 49 kDa proteins were enhanced, and encystment induction was promoted. Enzyme immunoassays (EIAs) showed that intracellular cAMP concentration was raised prior to encystment when the cells were stimulated by overpopulation. These results suggest that cAMP/PKA-dependent protein phosphorylation, which is an event on Ca²⁺-triggered signaling pathways, may be involved in encystment induction.     

Modeling of molecular and cellular mechanisms involved in Ca2+ signal encoding in airway myocytes

In airway myocytes signal transduction via cytosolic calcium plays an important role. In relation with experimental results we review models of basic molecular and cellular mechanisms involved in the signal transduction from the myocyte stimulation to the activation of the contractile apparatus. We concentrate on mechanisms for encoding of input signals into Ca²⁺ signals and the mechanisms for their decoding. The mechanisms are arranged into a general scheme of cellular signaling, the so-called bow-tie architecture of signaling, in which calcium plays the role of a common media for cellular signals and links the encoding and decoding part. The encoding of calcium signals in airway myocytes is better known and is presented in more detail. In particular, we focus on three recent models taking into account the intracellular calcium handling and ion fluxes through the plasma membrane. The model of membrane conductances was originally proposed for predicting membrane depolarization and voltage-dependent Ca²⁺ influx triggered by initial cytosolic Ca²⁺ increase as observed on cholinergic stimulation. Cellular models of intracellular Ca²⁺ handling were developed to investigate the role of a mixed population of InsP₃ receptor isoforms and the cellular environment in the occurrence of Ca²⁺ oscillations, and the respective role of the sarcoplasmic reticulum, mitochondria, and cytosolic Ca²⁺-binding proteins in cytosolic Ca²⁺ clearance. Modeling the mechanisms responsible for the decoding of calcium signals is developed in a lesser extent; however, the most recent theoretical studies are briefly presented in relation with the known experimental results.     

Identification of possible signal transduction components mediating light induction of the Gsa gene for an early chlorophyll biosynthetic step in Chlamydomonas reinhardtii

 Light-induced expression of the Gsa gene encoding the heme and chlorophyll biosynthetic enzyme glutamate 1-semialdehyde aminotransferase in Chlamydomonas reinhardtii was previously shown to involve Ca²⁺ and calmodulin (CaM) (C. lm et al. 1996, Plant Cell 8: 2245–2253). To further analyze the signal transduction pathway for light-induced Gsa expression, the effects of several pharmacological agents were examined. Treatment of light-dark synchronized cells with the heterotrimeric G-protein agonist Mas-7 caused partial induction of Gsa in the dark. The phospholipase C inhibitor U73122 inhibited light induction of Gsa. Exposure of cells to light caused a sustained 3-fold increase in cellular d-inositol 1,4,5-trisphosphate (InsP₃) concentration. KN-93, a specific inhibitor of Ca²⁺/CaM-dependent protein kinase II, inhibited light induction of Gsa. In contrast, cyclosporin A, a specific inhibitor of the Ca²⁺/CaM-dependent phosphoprotein phosphatase calcineurin, did not affect light induction of Gsa. These results, together with the earlier results, suggest the involvement of a canonical signal transduction pathway for light-regulated Gsa expression that involves a heterotrimeric G-protein activation, phospholipase C-catalyzed InsP₃ formation, InsP₃-dependent Ca²⁺ release, and activation of a downstream signaling pathway through a Ca²⁺/CaM-dependent protein kinase. Received: 21 October 1999 / Accepted: 3 December 1999     

Regulation of keratinocyte expression of stress proteins and antioxidants by the electrophilic nitrofatty acids 9- and 10-nitrooleic acid

Nitric oxide and various by-products including nitrite contribute to tissue injury by forming novel intermediates via redox-mediated nitration reactions. Nitration of unsaturated fatty acids generates electrophilic nitrofatty acids such as 9-nitrooleic acid (9-NO) and 10-nitrooleic acid (10-NO), which are known to initiate intracellular signaling pathways. In these studies, we characterized nitrofatty acid-induced signaling and stress protein expression in mouse keratinocytes. Treatment of keratinocytes with 5–25 μM 9-NO or 10-NO for 6 h upregulated mRNA expression of heat shock proteins (hsp's) 27 and 70; primary antioxidants heme oxygenase-1 (HO-1) and catalase; secondary antioxidants glutathione S-transferase (GST) A1/2, GSTA3, and GSTA4; and Cox-2, a key enzyme in prostaglandin biosynthesis. The greatest responses were evident with HO-1, hsp27, and hsp70. In keratinocytes, 9-NO activated JNK and p38 MAP kinases. JNK inhibition suppressed 9-NO-induced HO-1, hsp27, and hsp70 mRNA and protein expression, whereas p38 MAP kinase inhibition suppressed HO-1. In contrast, inhibition of constitutive expression of Erk1/2 suppressed only hsp70, indicating that 9-NO modulates expression of stress proteins by distinct mechanisms. 9-NO and 10-NO also upregulated expression of caveolin-1, the major structural component of caveolae. Western blot analysis of caveolar membrane fractions isolated by sucrose density centrifugation revealed that HO-1, hsp27, and hsp70 were localized within caveolae after nitrofatty acid treatment of keratinocytes, suggesting a link between induction of stress response proteins and caveolin-1 expression. These data indicate that nitrofatty acids are effective signaling molecules in keratinocytes. Moreover, caveolae seem to be important in the localization of stress proteins in response to nitrofatty acids.     

Human Golgi Antiapoptotic Protein Modulates Intracellular Calcium Fluxes

Golgi antiapoptotic protein (GAAP) is a novel regulator of cell death that is highly conserved in eukaryotes and present in some poxviruses, but its molecular mechanism is unknown. Given that alterations in intracellular Ca²⁺ homeostasis play an important role in determining cell sensitivity to apoptosis, we investigated if GAAP affected Ca²⁺ signaling. Overexpression of human (h)-GAAP suppressed staurosporine-induced, capacitative Ca²⁺ influx from the extracellular space. In addition, it reduced histamine-induced Ca²⁺ release from intracellular stores through inositol trisphosphate receptors. h-GAAP not only decreased the magnitude of the histamine-induced Ca²⁺ fluxes from stores to cytosol and mitochondrial matrices, but it also reduced the induction and frequency of oscillatory changes in cytosolic Ca²⁺. Overexpression of h-GAAP lowered the Ca²⁺ content of the intracellular stores and decreased the efficacy of IP₃, providing possible explanations for the observed results. Opposite effects were obtained when h-GAAP was knocked down by siRNA. Thus, our data demonstrate that h-GAAP modulates intracellular Ca²⁺ fluxes induced by both physiological and apoptotic stimuli.     

Mitochondria adjust Ca signaling regime to a pattern of stimulation in salivary acinar cells

The salivary acinar cells have unique Ca²⁺ signaling machinery that ensures an extensive secretion. The agonist-induced secretion is governed by Ca²⁺ signals originated from the endoplasmic reticulum (ER) followed by a store-operated Ca²⁺ entry (SOCE). During tasting and chewing food a frequency of parasympathetic stimulation increases up to ten fold, entailing cells to adapt its Ca²⁺ machinery to promote ER refilling and ensure sustained SOCE by yet unknown mechanism. By employing a combination of fluorescent Ca²⁺ imaging in the cytoplasm and inside cellular organelles (ER and mitochondria) we described the role of mitochondria in adjustment of Ca²⁺ signaling regime and ER refilling according to a pattern of agonist stimulation. Under the sustained stimulation, SOCE is increased proportionally to the degree of ER depletion. Cell adapts its Ca²⁺ handling system directing more Ca²⁺ into mitochondria via microdomains of high [Ca²⁺] providing positive feedback on SOCE while intra-mitochondrial tunneling provides adequate ER refilling. In the absence of an agonist, the bulk of ER refilling occurs through Ca²⁺-ATPase-mediated Ca²⁺ uptake within subplasmalemmal space. In conclusion, mitochondria play a key role in the maintenance of sustained SOCE and adequate ER refilling by regulating Ca²⁺ fluxes within the cell that may represent an intrinsic adaptation mechanism to ensure a long-lasting secretion.     

Mitochondria and calcium signaling in embryonic development

Calcium (Ca²⁺) is a simple but critical signal for controlling various cellular processes and is especially important in fertilization and embryonic development. The dynamic change of cellular Ca²⁺ concentration and homeostasis are tightly regulated. Cellular Ca²⁺ increases by way of Ca²⁺ influx from extracellular medium and Ca²⁺ release from cellular stores of the endoplasmic reticulum (ER) and sarcoplasmic reticulum (SR). The elevated Ca²⁺ is subsequently sequestered by expelling it out of the cell or by pumping back to the ER/SR. Mitochondria function as a power house for energy production via oxidative phosphorylation in most eukaryotes. In addition to this well-known function, mitochondria are also recognized to regulate Ca²⁺ homeostasis through different mechanisms. Although critical roles of Ca²⁺ signaling in fertilization and embryonic development are known, the involvement of mitochondria in these processes are not fully understood. This review is focused on the role of mitochondrial respiratory chain complex I in the regulation of Ca²⁺ signaling pathway and gene expression in embryonic development, especially on the new findings in the cardiac development of Xenopus embryos. The data demonstrate that mitochondria modulate Ca²⁺ signaling and the Ca²⁺-dependent NFAT pathway and its target gene which are essential for embryonic heart development.     

BAPTA-AM,an intracellular calcium chelator,inhibits RANKL-induced bone marrow macrophages differentiation through MEK/ERK,p38 MAPK and Akt,but not JNK pathways

To examine the roles of intracellular calcium in RANKL-induced bone marrow macrophages (BMMs) differentiation, the effects of intracellular calcium chelator BAPTA-AM on RANKL-induced BMMs differentiation, and the activation of its relating signal proteins (MAPKs, and the PI3K/Akt) were studied. BMMs were cultured with various concentrations of BAPTA-AM in the presence of M-CSF (25 ng/ml) and RANKL (25 ng/ml) for 7 days, osteoclastogenic ability, cytosolic free Ca²⁺ concentration, osteoclast survival and the expression of phosphorylated ERK1/2, SAPK/JNK, Akt and p38 MAPK were measured by TRAP staining, spectrofluorometer and Western blotting. BAPTA-AM inhibited osteoclastogenesis and osteoclast survival of BMMs by RANKL induction. In osteoclasts without the pretreatment of BAPTA-AM, the increased response of [Ca²⁺]_i was observed within 15 min and the maximum was about 1.2 times that of control. This response was sustained for 30 min and returned to the control level at 1 h after RANKL-inducing, and the increased response of [Ca²⁺]_i was completely abolished and sustained to at least 8 h by BAPTA-AM. Although immunoblotting data revealed that RANKL could activate the phosphorylation of ERK1/2, SAPK/JNK, Akt and p38 MAPK, the expression of ERK1/2, Akt and p38 MAPK phosphorylation was inhibited by BAPTA-AM dose-dependently. These results revealed that BAPTA-AM inhibit osteoclastogenic ability of BMMs via suppressing the increase of [Ca²⁺]_i which lead to inhibit RANKL-induced the phosphorylation of ERK, Akt and p38 MAPK, but not JNK. This finding may be useful in the development of an osteoclastic inhibitor that targets intracellular signaling factors.     

Differential Involvement of Intracellular Ca2+ in 1-Methyl-4-phenylpyridinium- or 6-Hydroxydopamine-Induced Cell Viability Loss in PC12 Cells

1-Methyl-4-phenylpyridinium (MPP⁺) or 6-hydroxydopamine (6-OHDA) caused a nuclear damage, the mitochondrial membrane permeability changes, leading to the cytochrome c release and caspase-3 activation, the formation of reactive oxygen species and the depletion of GSH in PC12 cells. Nicardipine (a calcium channel blocker), EGTA (an extracellular calcium chelator), BAPTA-AM (a cell permeable calcium chelator) and calmodulin antagonists (W-7 and calmidazolium) attenuated the MPP⁺-induced mitochondrial damage and cell death. In contrast, the compounds did not reduce the toxicity of 6-OHDA. Treatment with MPP⁺ or 6-OHDA evoked the elevation of intracellular Ca²⁺ levels. Unlike cell injury, addition of nicardipine, BAPTA-AM and calmodulin antagonists prevented the elevation of intracellular Ca²⁺ levels due to both toxins. The results show that the MPP⁺-induced formation of the mitochondrial permeability transition seems to be mediated by elevation of intracellular Ca²⁺ levels and calmodulin action. In contrast, the 6-OHDA-induced cell death seems to be mediated by Ca²⁺-independent manner.     

Calreticulin regulates TGF-β1-induced epithelial mesenchymal transition through modulating Smad signaling and calcium signaling

As a Ca²⁺ binding protein, calreticulin (CRT) has many functions and plays an important role in a variety of tumors. The role of CRT in TGF-β1-induced EMT is unknown. In this study, we demonstrated in vitro that TGF-β1-induced EMT elevated the expression of CRT in A549 lung cancer cells. Subsequently, we confirmed that overexpression CRT had no capacity to induce A549 cells EMT alone, but successfully enhanced TGF-β1-induced-EMT. Furthermore, knockdown of CRT in A549 cells significantly suppressed changes of EMT marks expression induced by TGF-β1. On treatment with TGF-β1, overexpression of CRT could enhance the phosphorylation of both Smad2 and Smad3. Consistently, the knockdown of CRT by siRNA-CRT could inhibit Smad signaling pathway activated by TGF-β1. These results indicated that CRT regulates EMT induced by TGF-β1 through Smad signaling pathway. Finally, TGF-β1-induced-EMT enhanced store-operated Ca²⁺ influx in A549 cells. CRT knockdown was able to abolish the effect of TGF-β1 on thapsigargin (TG) −induced Ca²⁺ release, but had failed to reduce store-operated Ca²⁺ influx. The alteration of intracellular Ca²⁺ concentration by TG or BAPTA-AM was able to regulate EMT induced by TGF-β1 through Smad signaling pathway. Together, these data identify that CRT regulates TGF-β1-induced-EMT through modulating Smad signaling. Furthermore, TGF-β1-induced-EMT is highly calcium-dependent, CRT was partly involved in it.     

Molecular and functional profiling of histamine receptor-mediated calcium ion signals in different cell lines

Calcium ions (Ca²⁺) play a pivotal role in cellular physiology. Often Ca²⁺-dependent processes are studied in commonly available cell lines. To induce Ca²⁺ signals on demand, cells may need to be equipped with additional proteins. A prominent group of membrane proteins evoking Ca²⁺ signals are G-protein coupled receptors (GPCRs). These proteins register external signals such as photons, odorants, and neurotransmitters and convey ligand recognition into cellular responses, one of which is Ca²⁺ signaling. To avoid receptor cross-talk or cross-activation with introduced proteins, the repertoire of cell-endogenous receptors must be known. Here we examined the presence of histamine receptors in six cell lines frequently used as hosts to study cellular signaling processes. In a concentration-dependent manner, histamine caused a rise in intracellular Ca²⁺ in HeLa, HEK 293, and COS-1 cells. The concentration for half-maximal activation (EC₅₀) was in the low micromolar range. In individual cells, transient Ca²⁺ signals and Ca²⁺ oscillations were uncovered. The results show that (i) HeLa, HEK 293, and COS-1 cells express sufficient amounts of endogenous receptors to study cellular Ca²⁺ signaling processes directly and (ii) these cell lines are suitable for calibrating Ca²⁺ biosensors in situ based on histamine receptor evoked responses.