Kirsten Welkenhuyzen

Ins(1,4,5)P3 receptor-mediated Ca2+ signaling and autophagy induction are interrelated

The role of intracellular Ca2+ signaling in starvation-induced autophagy remains unclear. Here, we examined Ca2+ dynamics during starvation-induced autophagy and the underlying molecular mechanisms. Tightly correlating with autophagy stimulation, we observed a remodeling of the Ca2+ signalosome. First, short periods of starvation (1 to 3 h) caused a prominent increase of the ER Ca2+-store content and enhanced agonist-induced Ca2+ release. The mechanism involved the upregulation of intralumenal ER Ca2+-binding proteins, calreticulin and Grp78/BiP, which increased the ER Ca2+-buffering capacity and reduced the ER Ca2+ leak. Second, starvation led to Ins(1,4,5)P3R sensitization. Immunoprecipitation experiments showed that during starvation Beclin 1, released from Bcl-2, first bound with increasing efficiency to Ins(1,4,5)P3Rs; after reaching a maximal binding after 3 h, binding, however, decreased again. The interaction site of Beclin 1 was determined to be present in the N-terminal Ins(1,4,5)P3-binding domain of the Ins(1,4,5)P3R. The starvation-induced Ins(1,4,5)P3R sensitization was abolished in cells treated with BECN1 siRNA, but not with ATG5 siRNA, pointing toward an essential role of Beclin 1 in this process. Moreover, recombinant Beclin 1 sensitized Ins(1,4,5)P3Rs in 45Ca2+-flux assays, indicating a direct regulation of Ins(1,4,5)P3R activity by Beclin 1. Finally, we found that Ins(1,4,5)P3R-mediated Ca2+ signaling was critical for starvation-induced autophagy stimulation, since the Ca2+ chelator BAPTA-AM as well as the Ins(1,4,5)P3R inhibitor xestospongin B abolished the increase in LC3 lipidation and GFP-LC3-puncta formation. Hence, our results indicate a tight and essential interrelation between intracellular Ca2+ signaling and autophagy stimulation as a proximal event in response to starvation.

mTOR-Controlled Autophagy Requires Intracellular Ca2+ Signaling

Autophagy is a lysosomal degradation pathway important for cellular homeostasis and survival. Inhibition of the mammalian target of rapamycin (mTOR) is the best known trigger for autophagy stimulation. In addition, intracellular Ca2+ regulates autophagy, but its exact role remains ambiguous. Here, we report that the mTOR inhibitor rapamycin, while enhancing autophagy, also remodeled the intracellular Ca2+-signaling machinery. These alterations include a) an increase in the endoplasmic-reticulum (ER) Ca2+-store content, b) a decrease in the ER Ca2+-leak rate, and c) an increased Ca2+ release through the inositol 1,4,5-trisphosphate receptors (IP3Rs), the main ER-resident Ca2+-release channels. Importantly, buffering cytosolic Ca2+ with BAPTA impeded rapamycin-induced autophagy. These results reveal intracellular Ca2+ signaling as a crucial component in the canonical mTOR-dependent autophagy pathway.

IP3R2 levels dictate the apoptotic sensitivity of diffuse large B-cell lymphoma cells to an IP3R-derived peptide targeting the BH4 domain of Bcl-2

Disrupting inositol 1,4,5-trisphosphate (IP3) receptor (IP3R)/B-cell lymphoma 2 (Bcl-2) complexes using a cell-permeable peptide (stabilized TAT-fused IP3R-derived peptide (TAT-IDPS)) that selectively targets the BH4 domain of Bcl-2 but not that of B-cell lymphoma 2-extra large (Bcl-Xl) potentiated pro-apoptotic Ca2+ signaling in chronic lymphocytic leukemia cells. However, the molecular mechanisms rendering cancer cells but not normal cells particularly sensitive to disrupting IP3R/Bcl-2 complexes are poorly understood. Therefore, we studied the effect of TAT-IDPS in a more heterogeneous Bcl-2-dependent cancer model using a set of ‘primed to death’ diffuse large B-cell lymphoma (DL-BCL) cell lines containing elevated Bcl-2 levels. We discovered a large heterogeneity in the apoptotic responses of these cells to TAT-IDPS with SU-DHL-4 being most sensitive and OCI-LY-1 being most resistant. This sensitivity strongly correlated with the ability of TAT-IDPS to promote IP3R-mediated Ca2+ release. Although total IP3R-expression levels were very similar among SU-DHL-4 and OCI-LY-1, we discovered that the IP3R2-protein level was the highest for SU-DHL-4 and the lowest for OCI-LY-1. Strikingly, TAT-IDPS-induced Ca2+ rise and apoptosis in the different DL-BCL cell lines strongly correlated with their IP3R2-protein level, but not with IP3R1-, IP3R3- or total IP3R-expression levels. Inhibiting or knocking down IP3R2 activity in SU-DHL-4-reduced TAT-IDPS-induced apoptosis, which is compatible with its ability to dissociate Bcl-2 from IP3R2 and to promote IP3-induced pro-apoptotic Ca2+ signaling. Thus, certain chronically activated B-cell lymphoma cells are addicted to high Bcl-2 levels for their survival not only to neutralize pro-apoptotic Bcl-2-family members but also to suppress IP3R hyperactivity. In particular, cancer cells expressing high levels of IP3R2 are addicted to IP3R/Bcl-2 complex formation and disruption of these complexes using peptide tools results in pro-apoptotic Ca2+ signaling and cell death.

Polycystin-1 and polycystin-2 are both required to amplify inositol-trisphosphate-induced Ca2+ release

Autosomal dominant polycystic kidney disease is caused by loss-of-function mutations in the PKD1 or PKD2 genes encoding respectively polycystin-1 and polycystin-2. Polycystin-2 stimulates the inositol trisphosphate (IP(3)) receptor (IP(3)R), a Ca(2+)-release channel in the endoplasmic reticulum (ER). The effect of ER-located polycystin-1 is less clear. Polycystin-1 has been reported both to stimulate and to inhibit the IP(3)R. We now studied the effect of polycystin-1 and of polycystin-2 on the IP(3)R activity under conditions where the cytosolic Ca(2+) concentration was kept constant and the reuptake of released Ca(2+) was prevented. We also studied the interdependence of the interaction of polycystin-1 and polycystin-2 with the IP(3)R. The experiments were done in conditionally immortalized human proximal-tubule epithelial cells in which one or both polycystins were knocked down using lentiviral vectors containing miRNA-based short hairpins. The Ca(2+) release was induced in plasma membrane-permeabilized cells by various IP(3) concentrations at a fixed Ca(2+) concentration under unidirectional (45)Ca(2+)-efflux conditions. We now report that knock down of polycystin-1 or of polycystin-2 inhibited the IP(3)-induced Ca(2+) release. The simultaneous presence of the two polycystins was required to fully amplify the IP(3)-induced Ca(2+) release, since the presence of polycystin-1 alone or of polycystin-2 alone did not result in an increased Ca(2+) release. These novel findings indicate that ER-located polycystin-1 and polycystin-2 operate as a functional complex. They are compatible with the view that loss-of-function mutations in PKD1 and in PKD2 both cause autosomal dominant polycystic kidney disease.

Resveratrol-induced autophagy is dependent on IP3Rs and on cytosolic Ca2 +☆

Previous work revealed that intracellular Ca2+ signals and the inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) are essential to increase autophagic flux in response to mTOR inhibition, induced by either nutrient starvation or rapamycin treatment. Here, we investigated whether autophagy induced by resveratrol, a polyphenolic phytochemical reported to trigger autophagy in a non-canonical way, also requires IP3Rs and Ca2+ signaling. Resveratrol augmented autophagic flux in a time-dependent manner in HeLa cells. Importantly, autophagy induced by resveratrol (80μM, 2h) was completely abolished in the presence of 10μM BAPTA-AM, an intracellular Ca2+-chelating agent. To elucidate the IP3Rs role in this process, we employed the recently established HEK 3KO cells lacking all three IP3R isoforms. In contrast to the HEK293 wt cells and to HEK 3KO cells re-expressing IP3R1, autophagic responses in HEK 3KO cells exposed to resveratrol were severely impaired. These altered autophagic responses could not be attributed to alterations in the mTOR/p70S6K pathway, since resveratrol-induced inhibition of S6 phosphorylation was not abrogated by chelating cytosolic Ca2+ or by knocking out IP3Rs. Finally, we investigated whether resveratrol by itself induced Ca2+ release. In permeabilized HeLa cells, resveratrol neither affected the sarco- and endoplasmic reticulum Ca2+ ATPase (SERCA) activity nor the IP3-induced Ca2+ release nor the basal Ca2+ leak from the ER. Also, prolonged (4 h) treatment with 100μM resveratrol did not affect subsequent IP3-induced Ca2+ release. However, in intact HeLa cells, although resveratrol did not elicit cytosolic Ca2+ signals by itself, it acutely decreased the ER Ca2+-store content irrespective of the presence or absence of IP3Rs, leading to a dampened agonist-induced Ca2+ signaling. In conclusion, these results reveal that IP3Rs and cytosolic Ca2+ signaling are fundamentally important for driving autophagic flux, not only in response to mTOR inhibition but also in response to non-canonical autophagy inducers like resveratrol. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Bax Inhibitor-1-mediated Ca2+ leak is decreased by cytosolic acidosis

Bax Inhibitor-1 (BI-1) is an evolutionarily conserved six-transmembrane domain endoplasmic reticulum (ER)-localized protein that protects against ER stress-induced apoptotic cell death. This function is closely connected to its ability to lower steady-state ER Ca2+ levels. Recently, we elucidated BI-1s Ca(2+)-channel pore in the C-terminal part of the protein and identified the critical amino acids of its pore. Based on these insights, a Ca(2+)-channel pore-dead mutant BI-1 (BI-1(D213R)) was developed. We determined whether BI-1 behaves as a bona fide H+/Ca2+ antiporter or as an ER Ca(2+)-leak channel by investigating the effect of pH on unidirectional Ca(2+)-efflux rates. At pH 6.8, wild-type BI-1 expression in BI-1(-/-) cells increased the ER Ca(2+)-leak rate, correlating with its localization in the ER compartment. In contrast, BI-1(D231R) expression in BI-1(-/-), despite its ER localization, did not increase the ER Ca(2+)-leak rate. However, at pH < 6.8, the BI-1-mediated ER Ca2+ leak was blocked. Finally, a peptide representing the Ca(2+)-channel pore of BI-1 promoting Ca2+ flux from the ER was used. Lowering the pH from 6.8 to 6.0 completely abolished the ability of the BI-1 peptide to mediate Ca2+ flux from the ER. We propose that this pH dependence is due to two aspartic acid residues critical for the function of the Ca(2+)-channel pore and located in the ER membrane-dipping domain, which facilitates the protonation of these residues.

The trans-membrane domain of Bcl-2α, but not its hydrophobic cleft, is a critical determinant for efficient IP 3 receptor inhibition

The anti-apoptotic Bcl-2 protein is emerging as an efficient inhibitor of IP3R function, contributing to its oncogenic properties. Yet, the underlying molecular mechanisms remain not fully understood. Using mutations or pharmacological inhibition to antagonize Bcl-2s hydrophobic cleft, we excluded this functional domain as responsible for Bcl-2-mediated IP3Rs inhibition. In contrast, the deletion of the C-terminus, containing the trans-membrane domain, which is only present in Bcl-2α, but not in Bcl-2β, led to impaired inhibition of IP3R-mediated Ca2+ release and staurosporine-induced apoptosis. Strikingly, the trans-membrane domain was sufficient for IP3R binding and inhibition. We therefore propose a novel model, in which the Bcl-2s C-terminus serves as a functional anchor, which beyond mere ER-membrane targeting, underlies efficient IP3R inhibition by (i) positioning the BH4 domain in the close proximity of its binding site on IP3R, thus facilitating their interaction; (ii) inhibiting IP3R-channel openings through a direct interaction with the C-terminal region of the channel downstream of the channel-pore. Finally, since the hydrophobic cleft of Bcl-2 was not involved in IP3R suppression, our findings indicate that ABT-199 does not interfere with IP3R regulation by Bcl-2 and its mechanism of action as a cell-death therapeutic in cancer cells likely does not involve Ca2+ signaling.

Polycystin-1 but not polycystin-2 deficiency causes upregulation of the mTOR pathway and can be synergistically targeted with rapamycin and metformin

Autosomal dominant polycystic kidney disease (ADPKD) is caused by loss-of-function mutations in either PKD1 or PKD2 genes, which encode polycystin-1 (TRPP1) and polycystin-2 (TRPP2), respectively. Increased activity of the mammalian target of rapamycin (mTOR) pathway has been shown in PKD1 mutants but is less documented for PKD2 mutants. Clinical trials using mTOR inhibitors were disappointing, while the AMP-activated kinase (AMPK) activator, metformin is not yet tested in patients. Here, we studied the mTOR activity and its upstream pathways in several human and mouse renal cell models with either siRNA or stable knockdown and with overexpression of TRPP2. Our data reveal for the first time differences between TRPP1 and TRPP2 deficiency. In contrast to TRPP1 deficiency, TRPP2-deficient cells did neither display excessive activation of the mTOR-kinase complex nor inhibition of AMPK activity, while ERK1/2 and Akt activity were similarly affected among TRPP1- and TRPP2-deficient cells. Furthermore, cell proliferation was more pronounced in TRPP1 than in TRPP2-deficient cells. Interestingly, combining low concentrations of rapamycin and metformin was more effective for inhibiting mTOR complex 1 activity in TRPP1-deficient cells than either drug alone. Our results demonstrate a synergistic effect of a combination of low concentrations of drugs suppressing the increased mTOR activity in TRPP1-deficient cells. This novel insight can be exploited in future clinical trials to optimize the efficiency and avoiding side effects of drugs in the treatment of ADPKD patients with PKD1 mutations. Furthermore, as TRPP2 deficiency by itself did not affect mTOR signaling, this may underlie the differences in phenotype, and genetic testing has to be considered for selecting patients for the ongoing trials.

HA14-1, but not the BH3 mimetic ABT-737, causes Ca2+ dysregulation in platelets and human cell lines.

Many Bcl-2-dependent cancer cells are primed to death due to their upregulation of BH3-only proteins in response to ongoing oncogenic stress. BH3-mimetic drugs like ABT-737 compete with Bim for binding to Bcl-2, releasing Bim and triggering Bax/Bak-mediated apoptosis.1 While ABT-737 causes regression of established tumors,2 it also limits platelet survival.3 The mechanisms underlying the observed thrombocytopenia have been the subject of debate. Since Bcl-Xl is essential for platelet life span,4 it was proposed that ABT-737, which does not discriminate between Bcl-2 and Bcl-Xl,5 kills platelets by antagonizing Bcl-Xl.3 However, these devastating effects of ABT-737 on platelet function were also associated with disturbed intracellular Ca2+ homeostasis and dynamics,3 but this remains poorly understood and controversial.6,7 We, therefore, explored the effect of ABT-737 on apoptosis in platelets and on intracellular Ca2+ signaling in platelets and human cell lines. We compared its effects on Ca2+ homeostasis with another Bcl-2 antagonist, the HA14-1 compound, which was reported to exert inhibitory properties on sarco/endoplasmic reticulum Ca2+-ATPases (SERCA).8 Washed platelets were prepared as described.9 For this, venous blood was collected from healthy donors. Permission was given by the Ethical Committee of the Leuven University Hospital to use blood from healthy individuals for further analysis. For apoptosis measurements, platelets were incubated with Annexin-V-FITC and analyzed with an Attune® Acoustic Focusing Flow Cytometer (Applied Biosystems). For the Ca2+ measurements in intact cells, platelets were seeded the day of measurement in poly-L-lysine-coated 96-well plates (Greiner) at a density of approximately 3 × 108 platelets/mL. Ca2+ measurements in intact Hela cells and unidirectional 45Ca2+-flux experiments in permeabilized cells were basically performed as previously described.10 SERCA2b ATPase activity was determined by colorimetric monitoring11 in a Ca2+-buffered solution containing microsomes (10 μg of protein) from HEK-293T cells ectopically expressing SERCA2b. Results are expressed as average ± standard deviation (SD). Significance was determined using two-tailed paired Students t-test. P<0.05 was considered significant. We confirmed that both HA14-1 (3 and 10 μM; 2 h) and ABT-737 (0.03, 0.1 and 0.3 μM; 2 h) provoked apoptosis in blood platelets (Figure 1A and B). However, only HA14-1, but not ABT-737, triggered a slow and steady increase in the cytosolic [Ca2+] originating from the intracellular Ca2+ stores in Fura2-loaded platelets exposed to extracellular EGTA (Figure 1C). Moreover, pre-treatment (10 μM; 30 min) of HA14-1, but not of ABT-737, reduced the total Ca2+ released from the ER in response to thapsigargin, an irreversible SERCA inhibitor, while not affecting store-operated Ca2+ influx (Figure 1D). To confirm our result in another cell model, we used the human cell line HeLa. Again, HA14-1, but not ABT-737, affected intracellular Ca2+ homeostasis in these cells in a concentration-dependent manner (Figure 2A). To investigate the underlying mechanism, we applied a highly quantitative 45Ca2+-flux assay10 in permeabilized HeLa cells to specifically assess ER 45Ca2+-uptake activity in the absence of plasmalemmal and mitochondrial Ca2+ fluxes and of IP3R-mediated Ca2+-release. Application of HA14-1 during the ER 45Ca2+-loading phase caused a strong decrease in the steady-state 45Ca2+ loading levels, while ABT-737 was much less effective (Figure 2B). The steady-state ER Ca2+ levels are determined by the balance between the ER Ca2+-uptake and ER Ca2+-leak activities. Importantly, application of either ABT-737 or HA14-1 (up to 30 μM) during the efflux phase did not affect the ER Ca2+-leak rate (Figure 2C), suggesting an inhibition of the ER Ca2+-uptake activity. Next, we directly assessed the effect of ABT-737 and HA14-1 on SERCA2b activity, the housekeeping isoform in human platelets,12 which was ectopically expressed in HEK-293T cells (Figure 2D). HA14-1 effectively inhibited SERCA2b Ca2+ ATPase activity (IC50 = 14 μM) while ABT-737 (up to 30 μM) had no effect. Finally, we assessed whether ABT-737 or HA14-1 directly affected IP3R-mediated Ca2+ release (e.g. which occurs in response to thrombin exposure of platelets). We used the 45Ca2+-flux assay to accurately measure IP3-induced Ca2+ release in permeabilized HeLa cells. We found that 30 μM HA14-1, but not ABT-737, inhibited the IP3R (Figure 2E). Performing a dose-response curve, we found that ABT-737 up to 100 μM did not inhibit IP3Rs. In contrast, HA14-1 potently inhibited IP3Rs at concentrations higher than 10 μM with an IC50 of approximately 50 mM and with an IP3R inhibition of approximately 80% at 100 μM (Figure 2F). Figure 1. The effect of HA14-1 and ABT-737 on apoptosis and intracellular Ca2+ signaling in platelets. (A-B) Flow-cytometry analysis of HA14-1 and ABT-737-induced apoptosis in platelets. (A) Histogram overlay representation of Annexin V-FITC staining of platelets ... Figure 2. The effect of HA14-1 and ABT-737 on ER Ca2+-uptake and –release mechanisms. (A) Fluorimetric analysis of the HA14-1 and ABT-737-induced Ca2+ responses in HeLa cells. The ratio of emitted fluorescence of Fura2 (excitation wavelength 340 nm/380 ... Our experiments clearly indicate that disrupted intracellular Ca2+ homeostasis is not a proximal event in ABT-737-induced thrombocytopenia. Thus, earlier studies indicating depleted intracellular Ca2+ homeostasis in platelets exposed for 10 μM ABT-737 for prolonged periods (e.g. 2 h) may reflect a late event that is the consequence of Bcl-Xl inhibition and ongoing cell death in platelets.6 We conclude that the dysregulation of intracellular Ca2+ signaling in platelets and human cell lines by HA14-1 is an off-target effect on SERCA2b and on IP3Rs, since on-target inhibition of Bcl-2/Bcl-Xl by ABT-737 does not disrupt intracellular Ca2+ signaling. Thus, ABT-737 has a safe Ca2+-signaling profile, since ABT-737, at therapeutically relevant concentrations (i.e. below 1 μM), does not affect intracellular Ca2+-transport mechanisms essential for cellular homeostasis.

Basal ryanodine receptor activity suppresses autophagic flux

Graphical abstract Figure. No Caption available. ABSTRACT The inositol 1,4,5‐trisphosphate receptors (IP3Rs) and intracellular Ca2+ signaling are critically involved in regulating different steps of autophagy, a lysosomal degradation pathway. The ryanodine receptors (RyR), intracellular Ca2+‐release channels mainly expressed in excitable cell types including muscle and neurons, have however not yet been extensively studied in relation to autophagy. Yet, aberrant expression and excessive activity of RyRs in these tissues has been implicated in the onset of several diseases including Alzheimer’s disease, where impaired autophagy regulation contributes to the pathology. In this study, we determined whether pharmacological RyR inhibition could modulate autophagic flux in ectopic RyR‐expressing models, like HEK293 cells and in cell types that endogenously express RyRs, like C2C12 myoblasts and primary hippocampal neurons. Importantly, RyR3 overexpression in HEK293 cells impaired the autophagic flux. Conversely, in all cell models tested, pharmacological inhibition of endogenous or ectopically expressed RyRs, using dantrolene or ryanodine, augmented autophagic flux by increasing lysosomal turn‐over (number of autophagosomes and autolysosomes measured as mCherry‐LC3 punctae/cell increased from 70.37 ± 7.81 in control HEK RyR3 cells to 111.18 ± 7.72 and 98.14 ± 7.31 after dantrolene and ryanodine treatments, respectively). Moreover, in differentiated C2C12 cells, transmission electron microscopy demonstrated that dantrolene treatment decreased the number of early autophagic vacuoles from 5.9 ± 2.97 to 1.8 ± 1.03 per cellular cross section. The modulation of the autophagic flux could be linked to the functional inhibition of RyR channels as both RyR inhibitors efficiently diminished the number of cells showing spontaneous RyR3 activity in the HEK293 cell model (from 41.14% ± 2.12 in control cells to 18.70% ± 2.25 and 9.74% ± 2.67 after dantrolene and ryanodine treatments, respectively). In conclusion, basal RyR‐mediated Ca2+‐release events suppress autophagic flux at the level of the lysosomes.