The regulation of neuronal mitochondrial metabolism by calcium.

Calcium signalling is fundamental to the function of the nervous system, in association with changes in ionic gradients across the membrane. Although restoring ionic gradients is energetically costly, a rise in intracellular Ca(2+) acts through multiple pathways to increase ATP synthesis, matching energy supply to demand. Increasing cytosolic Ca(2+) stimulates metabolite transfer across the inner mitochondrial membrane through activation of Ca(2+) -regulated mitochondrial carriers, whereas an increase in matrix Ca(2+) stimulates the citric acid cycle and ATP synthase. The aspartate-glutamate exchanger Aralar/AGC1 (Slc25a12), a component of the malate-aspartate shuttle (MAS), is stimulated by modest increases in cytosolic Ca(2+) and upregulates respiration in cortical neurons by enhancing pyruvate supply into mitochondria. Failure to increase respiration in response to small (carbachol) and moderate (K(+) -depolarization) workloads and blunted stimulation of respiration in response to high workloads (veratridine) in Aralar/AGC1 knockout neurons reflect impaired MAS activity and limited mitochondrial pyruvate supply. In response to large workloads (veratridine), acute stimulation of respiration occurs in the absence of MAS through Ca(2+) influx through the mitochondrial calcium uniporter (MCU) and a rise in matrix [Ca(2+) ]. Although the physiological importance of the MCU complex in work-induced stimulation of respiration of CNS neurons is not yet clarified, abnormal mitochondrial Ca(2+) signalling causes pathology. Indeed, loss of function mutations in MICU1, a regulator of MCU complex, are associated with neuromuscular disease. In patient-derived MICU1 deficient fibroblasts, resting matrix Ca(2+) is increased and mitochondria fragmented. Thus, the fine tuning of Ca(2+) signals plays a key role in shaping mitochondrial bioenergetics.

Reduction of endoplasmic reticulum Ca2+ levels favors plasma membrane surface exposure of calreticulin.

Some chemotherapeutic agents can elicit apoptotic cancer cell death, thereby activating an anticancer immune response that influences therapeutic outcome. We previously reported that anthracyclins are particularly efficient in inducing immunogenic cell death, correlating with the pre-apoptotic exposure of calreticulin (CRT) on the plasma membrane surface of anthracyclin-treated tumor cells. Here, we investigated the role of cellular Ca(2+) homeostasis on CRT exposure. A neuroblastoma cell line (SH-SY5Y) failed to expose CRT in response to anthracyclin treatment. This defect in CRT exposure could be overcome by the overexpression of Reticulon-1C, a manipulation that led to a decrease in the Ca(2+) concentration within the endoplasmic reticulum lumen. The combination of Reticulon-1C expression and anthracyclin treatment yielded more pronounced endoplasmic reticulum Ca(2+) depletion than either of the two manipulations alone. Chelation of intracellular (and endoplasmic reticulum) Ca(2+), targeted expression of the ligand-binding domain of the IP(3) receptor and inhibition of the sarco-endoplasmic reticulum Ca(2+)-ATPase pump reduced endoplasmic reticulum Ca(2+) load and promoted pre-apoptotic CRT exposure on the cell surface, in SH-SY5Y and HeLa cells. These results provide evidence that endoplasmic reticulum Ca(2+) levels control the exposure of CRT.

Regulation of autophagy by the inositol trisphosphate receptor.

The reduction of intracellular 1,4,5-inositol trisphosphate (IP(3)) levels stimulates autophagy, whereas the enhancement of IP(3) levels inhibits autophagy induced by nutrient depletion. Here, we show that knockdown of the IP(3) receptor (IP(3)R) with small interfering RNAs and pharmacological IP(3)R blockade is a strong stimulus for the induction of autophagy. The IP(3)R is known to reside in the membranes of the endoplasmic reticulum (ER) as well as within ER-mitochondrial contact sites, and IP(3)R blockade triggered the autophagy of both ER and mitochondria, as exactly observed in starvation-induced autophagy. ER stressors such as tunicamycin and thapsigargin also induced autophagy of ER and, to less extent, of mitochondria. Autophagy triggered by starvation or IP(3)R blockade was inhibited by Bcl-2 and Bcl-X(L) specifically targeted to ER but not Bcl-2 or Bcl-X(L) proteins targeted to mitochondria. In contrast, ER stress-induced autophagy was not inhibited by Bcl-2 and Bcl-X(L). Autophagy promoted by IP(3)R inhibition could not be attributed to a modulation of steady-state Ca(2+) levels in the ER or in the cytosol, yet involved the obligate contribution of Beclin-1, autophagy-related gene (Atg)5, Atg10, Atg12 and hVps34. Altogether, these results strongly suggest that IP(3)R exerts a major role in the physiological control of autophagy.

Mitochondrial dynamics and Ca2+ signaling.

Recent data shed light on two novel aspects of the mitochondria-Ca2+ liaison. First, it was extensively investigated how Ca2+ handling is controlled by mitochondrial shape, and positioning; a playground also of cell death and survival regulation. On the other hand, significant progress has been made to explore how intra- and near-mitochondrial Ca2+ signals modify mitochondrial morphology and cellular distribution. Here, we shortly summarize these advances and provide a model of Ca2+-mitochondria interactions.

Regulation of Ca2+ signalling and Ca2+-mediated cell death by the transcriptional coactivator PGC-1alpha.

Mitochondrial Ca2+ uptake controls cellular functions as diverse as aerobic metabolism, cytosolic Ca2+signalling and mitochondrial participation in apoptosis. Modulatory inputs converging on the organelle can regulate this process, determining the final outcome of Ca2+-mediated cell stimulation. We investigated in HeLa cells and primary skeletal myotubes the effect on Ca2+ signalling of the transcriptional peroxisome-proliferator-activated-receptor-gamma-coactivator-1alpha (PGC-1alpha), which triggers organelle biogenesis and modifies the mitochondrial proteome. PGC-1alpha selectively reduced mitochondrial Ca2+ responses to cell stimulation by reducing the efficacy of mitochondrial Ca2+ uptake sites and increasing organelle volume. In turn, this affected ER Ca2+ release and cytosolic responses in HeLa cells. Most importantly, the modulation of mitochondrial Ca2+ uptake significantly reduced cellular sensitivity to the Ca2+-mediated proapoptotic effect of C2 ceramide. These results reveal a primary role of PGC-1alpha in shaping mitochondrial participation in calcium signalling, that underlies its protective role against stress and proapoptotic stimuli in pathophysiological conditions.

Inositol 1,4,5-trisphosphate binding sites copurify with the putative Ca-storage protein calreticulin in rat liver.

Rat liver was homogenized and subjected to differential centrifugation. When the low speed nuclear pellet was processed on a Percoll gradient, plasma membrane markers and Ins(1,4,5)P3 binding activity purified together. The high speed (microsomal) fraction was subfractionated by sucrose density gradient centrifugation, resulting in 10-fold enrichment of [32P]-Ins(1,4,5)P3 binding. In the sucrose density gradient fractions there was an inverse relationship between the enrichment of plasma membrane markers and Ins(1,4,5)P3 binding sites. Endoplasmic reticulum markers showed a moderate enrichment in the fractions displaying high Ins(1,4,5)P3 binding activity. Calcium binding proteins in the homogenate and in the microsomal subfractions were separated by SDS/PAGE. A 60 kD protein, stained metachromatically with Stains-All was identified as calreticulin with immunoblotting. Its enrichment pattern was similar to that of Ins(1,4,5)P3 binding sites, indicating the co-existence of these two elements of Ca(2+)-metabolism in the same intracellular compartment in the liver.

Voltage dependent calcium channels in adrenal glomerulosa cells and in insulin producing cells.

We have examined the structure and function of Ca2+ channels in excitable endocrine cell types, in rat adrenal glomerulosa cells and in two insulin producing cell types, the rat pancreatic beta cell and the INS-1 cell line. In previous studies on glomerulosa cells, we observed low (T-type) and high threshold (L-type) voltage dependent Ca2+ currents in addition to a K+ induced inward rectifying Ca2+ current (Igl). beta cells are known to exhibit T-, L- and N-type currents. We have now found that INS-1 cells also show low threshold (T-type) and high threshold Ca2+ currents. The latter was further resolved by organic inhibitors into L-type and P/Q-type currents and no Igl was detected. The expression of the pore-forming alpha 1 subunit of voltage dependent Ca2+ channels was studied by means of reverse transcription-polymerase chain reaction (RT-PCR), followed by restriction enzyme mapping and/or sequencing. Both in glomerulosa and pancreatic beta cells, the neuroendocrine (D) class of the alpha 1 subunit, known to be responsible for L-type current, represents the majority of the PCR product. Comparable amounts of the neuroendocrine (D) and the neuronal A-type alpha 1 subunits dominate the message in INS-1 cells. Different characteristics of Ca2+ currents in these cell types is discussed in view of the channel repertoire.

Endoplasmic reticulum, Bcl-2 and Ca2+ handling in apoptosis.

In the complex signalling interplay that allows extracellular signals to be decoded into activation of apoptotic cell death, Ca(2+) plays a significant role. This is supported not only by evidence linking alterations in Ca(2+) homeostasis to the triggering of apoptotic (and in some cases necrotic) cell death, but also by recent data indicating that a key anti-apoptotic protein, Bcl-2, has a direct effect on ER Ca(2+) handling. We will briefly summarise the first aspect, and describe in more detail these new data, demonstrating that (i) Bcl-2 reduces the state of filling of the ER Ca(2+) store and (ii) this Ca(2+) signalling alteration renders the cells less sensitive to apoptotic stimuli. Overall, these results suggest that calcium homeostasis may represent a pharmacological target in the fundamental pathological process of apoptosis.

Inositol 1,4,5-trisphosphate receptor subtypes in adrenal glomerulosa cells.

Expression of the diverse subtypes of inositol 1,4,5-trisphosphate (InsP3) receptor (IP3R) was examined in rat adrenal glomerulosa cells. The polymerase chain reaction products were characterized by means of DNA sequencing and/or restriction enzyme mapping. The predominant subtype expressed is IP3R-1; its alternatively spliced variants containing and lacking segment S1 are present in comparable amounts. The expression level of IP3R-2 is about a quarter that of IP3R-1, whereas IP3R-3 is expressed at a very low level. Sodium depletion, a chronic physiological stimulus of glomerulosa cells, failed to influence the expression of IP3R-1, as measured by competitive polymerase chain reaction, and failed to modify the ratio of the different receptor subtypes, as studied with restriction enzyme mapping.

Selective inhibition of potassium-stimulated rat adrenal glomerulosa cells by ruthenium red.

The effect of the cationic dye, ruthenium red (RR), on ionic fluxes, Ca2+ signal generation, and stimulation of aldosterone production was studied in isolated rat adrenal glomerulosa cells. In these cells, increased extracellular [K+] as well as angiotensin II (Ang II) elevate cytoplasmic Ca2+ concentration and thereupon activate steroidogenesis. However, the mode of action of the two stimuli are different: while a dihidropyridine-sensitive mechanism contributes to the response to both agonists, Ang II induces Ca2+ release from intracellular stores and causes capacitative Ca2+ influx, whereas K+ was recently shown to activate a plasma membrane Ca2+ current (Igl) independently of membrane depolarization. The difference is reflected in the sensitivity of the response of the cells to RR. The Ang II-induced Ca2+ signal and aldosterone production were not inhibited, but rather slightly potentiated by the dye. This potentiation was probably the consequence of the membrane-depolarizing effect of RR, due to the observed inhibition of the resting K+ conductance. Conversely, Ca2+ signal and aldosterone production were significantly reduced by RR when the cells were stimulated by moderately elevated [K+] (6-8 mM). Our patch clamp studies suggest that this effect was related to the inhibition of different voltage-dependent and -independent inward Ca2+ currents and indicates the functional importance of the latter in the signal transduction of the potassium-stimulated glomerulosa cell.

Expression of inositol 1,4,5-trisphosphate receptors in rat adrenocortical zones.

Previously we demonstrated the presence of InsP3R-I, -II and -III subtypes in the zona glomerulosa. Now we have examined the expression of different subtypes of inositol 1,4,5-trisphosphate receptor (InsP3R) in the inner zones of rat adrenal cortex. RNA extracted from decapsulated adrenal tissue (zonae fasciculata-reticularis and the medulla) or from fasciculata-reticularis cells was reverse transcribed. Subsequent polymerase chain reaction revealed the presence of InsP3R-I, -II and -III subtypes in decapsulated tissue but failed to demonstrate the expression of any known subtypes of InsP3R in fasciculata-reticularis cells. Accordingly, InsP3 receptors expressed in the decapsulated tissue are of medullary origin.

Systems biology of cisplatin resistance: past, present and future.

The platinum derivative cis-diamminedichloroplatinum(II), best known as cisplatin, is currently employed for the clinical management of patients affected by testicular, ovarian, head and neck, colorectal, bladder and lung cancers. For a long time, the antineoplastic effects of cisplatin have been fully ascribed to its ability to generate unrepairable DNA lesions, hence inducing either a permanent proliferative arrest known as cellular senescence or the mitochondrial pathway of apoptosis. Accumulating evidence now suggests that the cytostatic and cytotoxic activity of cisplatin involves both a nuclear and a cytoplasmic component. Despite the unresolved issues regarding its mechanism of action, the administration of cisplatin is generally associated with high rates of clinical responses. However, in the vast majority of cases, malignant cells exposed to cisplatin activate a multipronged adaptive response that renders them less susceptible to the antiproliferative and cytotoxic effects of the drug, and eventually resume proliferation. Thus, a large fraction of cisplatin-treated patients is destined to experience therapeutic failure and tumor recurrence. Throughout the last four decades great efforts have been devoted to the characterization of the molecular mechanisms whereby neoplastic cells progressively lose their sensitivity to cisplatin. The advent of high-content and high-throughput screening technologies has accelerated the discovery of cell-intrinsic and cell-extrinsic pathways that may be targeted to prevent or reverse cisplatin resistance in cancer patients. Still, the multifactorial and redundant nature of this phenomenon poses a significant barrier against the identification of effective chemosensitization strategies. Here, we discuss recent systems biology studies aimed at deconvoluting the complex circuitries that underpin cisplatin resistance, and how their findings might drive the development of rational approaches to tackle this clinically relevant problem.

DJ-1 protects against cell death following acute cardiac ischemia-reperfusion injury.

Novel therapeutic targets are required to protect the heart against cell death from acute ischemia-reperfusion injury (IRI). Mutations in the DJ-1 (PARK7) gene in dopaminergic neurons induce mitochondrial dysfunction and a genetic form of Parkinson's disease. Genetic ablation of DJ-1 renders the brain more susceptible to cell death following ischemia-reperfusion in a model of stroke. Although DJ-1 is present in the heart, its role there is currently unclear. We sought to investigate whether mitochondrial DJ-1 may protect the heart against cell death from acute IRI by preventing mitochondrial dysfunction. Overexpression of DJ-1 in HL-1 cardiac cells conferred the following beneficial effects: reduced cell death following simulated IRI (30.4±4.7% with DJ-1 versus 52.9±4.7% in control; n=5, P<0.05); delayed mitochondrial permeability transition pore (MPTP) opening (a critical mediator of cell death) (260±33 s with DJ-1 versus 121±12 s in control; n=6, P<0.05); and induction of mitochondrial elongation (81.3±2.5% with DJ-1 versus 62.0±2.8% in control; n=6 cells, P<0.05). These beneficial effects of DJ-1 were absent in cells expressing the non-functional DJ-1(L166P) and DJ-1(Cys106A) mutants. Adult mice devoid of DJ-1 (KO) were found to be more susceptible to cell death from in vivo IRI with larger myocardial infarct sizes (50.9±3.5% DJ-1 KO versus 41.1±2.5% in DJ-1 WT; n≥7, P<0.05) and resistant to cardioprotection by ischemic preconditioning. DJ-1 KO hearts showed increased mitochondrial fragmentation on electron microscopy, although there were no differences in calcium-induced MPTP opening, mitochondrial respiratory function or myocardial ATP levels. We demonstrate that loss of DJ-1 protects the heart from acute IRI cell death by preventing mitochondrial dysfunction. We propose that DJ-1 may represent a novel therapeutic target for cardioprotection.

The protein disulfide isomerases PDIA4 and PDIA6 mediate resistance to cisplatin-induced cell death in lung adenocarcinoma.

Intrinsic and acquired chemoresistance are frequent causes of cancer eradication failure. Thus, long-term cis-diaminedichloroplatine(II) (CDDP) or cisplatin treatment is known to promote tumor cell resistance to apoptosis induction via multiple mechanisms involving gene expression modulation of oncogenes, tumor suppressors and blockade of pro-apoptotic mitochondrial membrane permeabilization. Here, we demonstrate that CDDP-resistant non-small lung cancer cells undergo profound remodeling of their endoplasmic reticulum (ER) proteome (>80 proteins identified by proteomics) and exhibit a dramatic overexpression of two protein disulfide isomerases, PDIA4 and PDIA6, without any alteration in ER-cytosol Ca(2+) fluxes. Using pharmacological and genetic inhibition, we show that inactivation of both proteins directly stimulates CDDP-induced cell death by different cellular signaling pathways. PDIA4 inactivation restores a classical mitochondrial apoptosis pathway, while knockdown of PDIA6 favors a non-canonical cell death pathway sharing some necroptosis features. Overexpression of both proteins has also been found in lung adenocarcinoma patients, suggesting a clinical importance of these proteins in chemoresistance.

PGC-1ß mediates adaptive chemoresistance associated with mitochondrial DNA mutations.

Primary mitochondrial dysfunction commonly leads to failure in cellular adaptation to stress. Paradoxically, however, nonsynonymous mutations of mitochondrial DNA (mtDNA) are frequently found in cancer cells and may have a causal role in the development of resistance to genotoxic stress induced by common chemotherapeutic agents, such as cis-diammine-dichloroplatinum(II) (cisplatin, CDDP). Little is known about how these mutations arise and the associated mechanisms leading to chemoresistance. Here, we show that the development of adaptive chemoresistance in the A549 non-small-cell lung cancer cell line to CDDP is associated with the hetero- to homoplasmic shift of a nonsynonymous mutation in MT-ND2, encoding the mitochondrial Complex-I subunit ND2. The mutation resulted in a 50% reduction of the NADH:ubiquinone oxidoreductase activity of the complex, which was compensated by increased biogenesis of respiratory chain complexes. The compensatory mitochondrial biogenesis was most likely mediated by the nuclear co-activators peroxisome proliferator-activated receptor gamma co-activator-1α (PGC-1α) and PGC-1ß, both of which were significantly upregulated in the CDDP-resistant cells. Importantly, both transient and stable silencing of PGC-1ß re-established the sensitivity of these cells to CDDP-induced apoptosis. Remarkably, the PGC-1ß-mediated CDDP resistance was independent of the mitochondrial effects of the co-activator. Altogether, our results suggest that partial respiratory chain defects because of mtDNA mutations can lead to compensatory upregulation of nuclear transcriptional co-regulators, in turn mediating resistance to genotoxic stress.

SCaMC-1 promotes cancer cell survival by desensitizing mitochondrial permeability transition via ATP/ADP-mediated matrix Ca(2+) buffering.

Ca(2+)-mediated mitochondrial permeability transition (mPT) is the final common pathway of stress-induced cell death in many major pathologies, but its regulation in intact cells is poorly understood. Here we report that the mitochondrial carrier SCaMC-1/SLC25A24 mediates ATP-Mg(2-)/Pi(2-) and/or HADP(2-)/Pi(2-) uptake into the mitochondria after an increase in cytosolic [Ca(2+)]. ATP and ADP contribute to Ca(2+) buffering in the mitochondrial matrix, resulting in desensitization of the mPT. Comprehensive gene expression analysis showed that SCaMC-1 overexpression is a general feature of transformed and cancer cells. Knockdown of the transporter led to vast reduction of mitochondrial Ca(2+) buffering capacity and sensitized cells to mPT-mediated necrotic death triggered by oxidative stress and Ca(2+) overload. These findings revealed that SCaMC-1 exerts a negative feedback control between cellular Ca(2+) overload and mPT-dependent cell death, suggesting that the carrier might represent a novel target for cancer therapy.

Review article: pancreatic renin-angiotensin systems in health and disease.

BACKGROUND: In addition to the circulating (endocrine) renin-angiotensin system (RAS), local renin-angiotensin systems are now known to exist in diverse cells and tissues. Amongst these, pancreatic renin-angiotensin systems have recently been identified and may play roles in the physiological regulation of pancreatic function, as well as being implicated in the pathogenesis of pancreatic diseases including diabetes, pancreatitis and pancreatic cancer. AIM: To review and summarise current knowledge of pancreatic renin-angiotensin systems. METHODS: We performed an extensive PubMed, Medline and online review of all relevant literature. RESULTS: Pancreatic RAS appear to play various roles in the regulation of pancreatic physiology and pathophysiology. Ang II may play a role in the development of pancreatic ductal adenocarcinoma, via stimulation of angiogenesis and prevention of chemotherapy toxicity, as well as in the initiation and propagation of acute pancreatitis (AP); whereas, RAS antagonism is capable of preventing new-onset diabetes and improving glycaemic control in diabetic patients. Current evidence for the roles of pancreatic RAS is largely based upon cell and animal models, whilst definitive evidence from human studies remains lacking. CONCLUSIONS: The therapeutic potential for RAS antagonism, using cheap and widely available agents, and may be untapped and such roles are worthy of active investigation in diverse pancreatic disease states.

Anabolic androgenic steroids and intracellular calcium signaling: a mini review on mechanisms and physiological implications.

Increasing evidence suggests that nongenomic effects of testosterone and anabolic androgenic steroids (AAS) operate concertedly with genomic effects. Classically, these responses have been viewed as separate and independent processes, primarily because nongenomic responses are faster and appear to be mediated by membrane androgen receptors, whereas long-term genomic effects are mediated through cytosolic androgen receptors regulating transcriptional activity. Numerous studies have demonstrated increases in intracellular Ca2+ in response to AAS. These Ca2+ mediated responses have been seen in a diversity of cell types, including osteoblasts, platelets, skeletal muscle cells, cardiac myocytes and neurons. The versatility of Ca2+ as a second messenger provides these responses with a vast number of pathophysiological implications. In cardiac cells, testosterone elicits voltage-dependent Ca2+ oscillations and IP3R-mediated Ca2+ release from internal stores, leading to activation of MAPK and mTOR signaling that promotes cardiac hypertrophy. In neurons, depending upon concentration, testosterone can provoke either physiological Ca2+ oscillations, essential for synaptic plasticity, or sustained, pathological Ca2+ transients that lead to neuronal apoptosis. We propose therefore, that Ca2+ acts as an important point of crosstalk between nongenomic and genomic AAS signaling, representing a central regulator that bridges these previously thought to be divergent responses.

The inositol 1,4,5-trisphosphate receptor regulates autophagy through its interaction with Beclin 1.

The inositol 1,4,5-trisphosphate receptor (IP(3)R) is a major regulator of apoptotic signaling. Through interactions with members of the Bcl-2 family of proteins, it drives calcium (Ca(2+)) transients from the endoplasmic reticulum (ER) to mitochondria, thereby establishing a functional and physical link between these organelles. Importantly, the IP(3)R also regulates autophagy, and in particular, its inhibition/depletion strongly induces macroautophagy. Here, we show that the IP(3)R antagonist xestospongin B induces autophagy by disrupting a molecular complex formed by the IP(3)R and Beclin 1, an interaction that is increased or inhibited by overexpression or knockdown of Bcl-2, respectively. An effect of Beclin 1 on Ca(2+) homeostasis was discarded as siRNA-mediated knockdown of Beclin 1 did not affect cytosolic or luminal ER Ca(2+) levels. Xestospongin B- or starvation-induced autophagy was inhibited by overexpression of the IP(3)R ligand-binding domain, which coimmunoprecipitated with Beclin 1. These results identify IP(3)R as a new regulator of the Beclin 1 complex that may bridge signals converging on the ER and initial phagophore formation.