Anne-Marie Vacher

CD95 triggers Orai1-mediated localized Ca2+ entry, regulates recruitment of protein kinase C (PKC) β2, and prevents death-inducing signaling complex formation

The death receptor CD95 plays a pivotal role in immune surveillance and immune tolerance. Binding of CD95L to CD95 leads to recruitment of the adaptor protein Fas-associated death domain protein (FADD), which in turn aggregates caspase-8 and caspase-10. Efficient formation of the CD95/FADD/caspase complex, known as the death-inducing signaling complex (DISC), culminates in the induction of apoptosis. We show that cells exposed to CD95L undergo a reorganization of the plasma membrane in which the Ca2+ release-activated Ca2+ channel Orai1 and the endoplasmic reticulum-resident activator stromal interaction molecule 1 colocalize with CD95 into a micrometer-sized cluster in which the channel elicits a polarized entry of calcium. Orai1 knockdown and expression of a dominant negative construct (Orai1E106A) reveal that on CD95 engagement, the Orai1-driven localized Ca2+ influx is fundamental to recruiting the Ca2+-dependent protein kinase C (PKC) β2 to the DISC. PKCβ2 in turn transiently holds the complex in an inactive status, preventing caspase activation and transmission of the apoptotic signal. This study identifies a biological role of Ca2+ and the Orai1 channel that drives a transient negative feedback loop, introducing a lag phase in the early steps of the CD95 signal. We suggest that these localized events provide a time of decision to prevent accidental cell death.

CD95-Mediated Calcium Signaling Promotes T Helper 17 Trafficking to Inflamed Organs in Lupus-Prone Mice

Summary CD95 ligand (CD95L) is expressed by immune cells and triggers apoptotic death. Metalloprotease-cleaved CD95L (cl-CD95L) is released into the bloodstream but does not trigger apoptotic signaling. Hence, the pathophysiological role of cl-CD95L remains unclear. We observed that skin-derived endothelial cells from systemic lupus erythematosus (SLE) patients expressed CD95L and that after cleavage, cl-CD95L promoted T helper 17 (Th17) lymphocyte transmigration across the endothelial barrier at the expense of T regulatory cells. T cell migration relied on a direct interaction between the CD95 domain called calcium-inducing domain (CID) and the Src homology 3 domain of phospholipase Cγ1. Th17 cells stimulated with cl-CD95L produced sphingosine-1-phosphate (S1P), which promoted endothelial transmigration by activating the S1P receptor 3. We generated a cell-penetrating CID peptide that prevented Th17 cell transmigration and alleviated clinical symptoms in lupus mice. Therefore, neutralizing the CD95 non-apoptotic signaling pathway could be an attractive therapeutic approach for SLE treatment.

Galanin evokes a cytosolic calcium bursting mode and hormone release in GH3/B6 pituitary cells

The effects of galanin on secretion and cytosolic free Ca2+ concentration ([Ca2+ i) have been studied in GH3/B6 pituitary cells. Prolactin (PRL) and growth hormone (GH) release was measured in column Perifusion experiments; [Ca2+]i was monitored in single cells by dual emission microspectrofluorimetry using indo‐1 as intracellular Ca2+ probe. Galanin (0.1–1 nM) caused PRL and GH release coincident with a modest rise in [Ca2+]i The increase in [Ca2+]i comprises the establishment of characteristic long‐lasting bursts of [Ca2+]i transients. Galanin acts on Ca2+ entry through voltage‐gated Ca2+ channels since there was no response to the peptide when Cd2+ ‐ a Ca2+ channel blocker ‐ was added to the bath solution. The stimulation of bursting activity by galanin may provide a fine Ca2+ ‐signalling mechanism which maximally stimulates hormone release while avoiding refractory periods.

Does calcium contribute to the CD95 signaling pathway

Death receptors play a crucial role in immune surveillance and cellular homeostasis, two processes circumvented by tumor cells. CD95 (also termed Fas or APO1) is a transmembrane receptor, which belongs to the tumor necrosis factor receptor superfamily, and induces a potent apoptotic signal. Initial steps of the CD95 signal take place through protein/protein interactions that bring zymogens such as caspase-8 and caspase-10 closer. Aggregation of these procaspases leads to their autoprocessing, to the release of activated caspases in the cytosol, which causes a caspase cascade, and to the transmission of the apoptotic signal. In parallel, CD95 engagement drives an increase in the intracellular calcium concentration (Ca2+)i whose origin and functions remain controversial. Although Ca2+ ions play a central role in apoptosis/necrosis induction, recent studies have highlighted a protective role of Ca2+ in death receptor signaling. In the light of these findings, we discuss the role of Ca2+ ions as modulators of CD95 signaling.

The antidepressant fluoxetine induces necrosis by energy depletion and mitochondrial calcium overload

Selective Serotonin Reuptake Inhibitor antidepressants, such as fluoxetine (Prozac), have been shown to induce cell death in cancer cells, paving the way for their potential use as cancer therapy. These compounds are able to increase cytosolic calcium concentration ([Ca2+]cyt), but the involved mechanisms and their physiological consequences are still not well understood. Here, we show that fluoxetine induces an increase in [Ca2+]cyt by emptying the endoplasmic reticulum (ER) through the translocon, an ER Ca2+ leakage structure. Our data also show that fluoxetine inhibits oxygen consumption and lowers mitochondrial ATP. This latter is essential for Ca2+ reuptake into the ER, and we postulated therefore that the fluoxetine-induced decrease in mitochondrial ATP production results in the emptying of the ER, leading to capacitative calcium entry. Furthermore, Ca2+ quickly accumulated in the mitochondria, leading to mitochondrial Ca2+ overload and cell death. We found that fluoxetine could induce an early necrosis in human peripheral blood lymphocytes and Jurkat cells, and could also induce late apoptosis, especially in the tumor cell line. These results shed light on fluoxetine-induced cell death and its potential use in cancer treatment.

Localized Store-Operated Calcium Influx Represses CD95-Dependent Apoptotic Effects of Rituximab in Non-Hodgkin B Lymphomas

The anti-CD20 mAb, rituximab, is routinely used to treat B cell malignancies. However, a majority of patients relapse. An improvement in the complete response was obtained by combining rituximab with chemotherapy, at the cost of increased toxicity. We reported that rituximab induced the colocalization of both the Orai1 Ca2+ release-activated Ca2+ channel (CRAC) and the endoplasmic reticulum Ca2+ sensor stromal interaction molecule 1 with CD20 and CD95 into a cluster, eliciting a polarized store-operated Ca2+ entry (SOCE). We observed that blocking this Ca2+ entry with downregulation of Orai1, pharmacological inhibitors, or reducing calcemia with hypocalcemic drugs sensitized human B lymphoma cell lines and primary human lymphoma cells to rituximab-induced apoptosis in vitro, and improved the antitumoral effect of rituximab in xenografted mice. This revealed that Ca2+ entry exerted a negative feedback loop on rituximab-induced apoptosis, suggesting that associating CRAC channel inhibitors or hypocalcemic agents with rituximab may improve the treatment of patients with B cell malignancies. The calcium-dependent proteins involved in this process appear to vary according to the B lymphoma cell type, suggesting that CRAC-channel targeting is likely to be more efficient than calcium-dependent protein targeting.

Prolactin and growth hormone release and calcium influx are stimulated by galanin within a 'Window' range of concentrations in pituitary GH3 B6 cells

Galanin is widely distributed throughout the rat neural and endocrine system with the highest concentration in the anterior pituitary. The effects of galanin were investigated in rat tumor pituitary cells (GH3/B6). A single stimulation with galanin caused prolactin (PRL) and growth hormone (GH) release within a narrow range of concentrations (10(-11)-10(-9) M). However, secretory responses were not consistently observed upon subsequent galanin stimulation. Galanin triggered a rise in cytosolic Ca2+ (burst in [Ca2+]i transients) with a similar responsiveness. By contrast, no change in PRL mRNA was detectable in response to the peptide. These data suggest that galanin which binds to high-affinity receptors in anterior pituitary cells can exert subtle modulations of pituitary cell functions.

Abstract 3723: Regulation of TRAIL-induced apoptotic signaling by the autophagy receptor p62 in acute promyelocytic leukemia cells

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Autophagy is an evolutionary conserved process that degrades and recycles cellular components through the lysosomal pathway to maintain cellular homeostasis. It has long been considered as a pro-survival mechanism to maintain viability under stressful conditions. However, a growing body of evidence demonstrates that autophagy could also promote or act as a pro-death program. Autophagy plays an ambivalent role in cancer: it can operate either as a pro-tumoral mechanism or paradoxically as an anti-tumoral mechanism depending on the context and the stage of the tumours, making it an interesting field of investigation. p62/SQSTM1 is an adapter protein playing a key role during the autophagy process. It promotes the selective degradation of ubiquitinated cellular substrates (i.e. proteins and organelles). As a multidomain protein adapter, p62 is involved in the regulation of various signaling pathways including those that control cell death pathways. In this regard, p62 can directly interact with the death receptor DR5 and promotes death receptor induced-apoptosis through caspase 8 stabilization. Acute myeloid leukemia (AML) is a heterogeneous disease characterized by an abnormal proliferation of myeloblasts (precursors of myeloid white blood cells) without differentiation. Acute promyelocytic leukemia (APL) is a subtype of AML characterized by the translocation t(15;17)(q22;q12) which results to the expression of the chimeric protein PML-RAR alpha. All-trans retinoic acid (ATRA) induces cancer cells differentiation and results in complete clinical remission in APL patients. It is now used as a first-line therapy. Despite the success of this treatment, some patients are refractory to ATRA or relapse. Thus, new therapeutic strategies are needed. We previously showed that ATRA-induced APL cell differentiation was associated with autophagy induction and p62/SQSMT1 accumulation, a response that confers a survival advantage to mature APL cells. As recent findings showed several molecular links between p62 and extrinsic apoptosis pathway, we decided to investigate the link between p62/SQSMT1 and cell death in AML cells upon treatment by TRAIL, an inducer of extrinsic apoptosis. We found that p62/SQSTM1 is required for apoptotic responses in APL cells. Our results also reveal the synergic cooperation between TRAIL and ATRA to increase apoptosis. We are currently investigating the molecular mechanisms underlying the role of autophagy as well as the autophagy receptor p62/SQSM1 in TRAIL-induced apoptosis in APL cells. Citation Format: Kelly Airiau, Olivier Micheau, Anne-Marie Vacher, Faten Mehri, Pierre Vacher, Mojgan Djavaheri-Mergny. Regulation of TRAIL-induced apoptotic signaling by the autophagy receptor p62 in acute promyelocytic leukemia cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3723.