Ricardo Armisen

Cell Death by Necrosis, a Regulated Way to Go

Apoptosis is a programmed form of cell death with well-defined morphological traits that are often associated with activation of caspases. More recently evidence has become available demonstrating that upon caspase inhibition alternative programs of cell death are executed, including ones with features characteristic of necrosis. These findings have changed our view of necrosis as a passive and essentially accidental form of cell death to that of an active, regulated and controllable process. Also necrosis has now been observed in parallel with, rather than as an alternative pathway to, apoptosis. Thus, cell death responses are extremely flexible despite being programmed. In this review, some of the hallmarks of different programmed cell death modes have been highlighted before focusing the discussion on necrosis. Obligatory events associated with this form of cell death include uncompensated cell swelling and related changes at the plasma membrane. In this context, representatives of the transient receptor channel family and their regulation are discussed. Also mechanisms that lead to execution of the necrotic cell death program are highlighted. Emphasis is laid on summarizing our understanding of events that permit switching between cell death modes and how they connect to necrosis. Finally, potential implications for the treatment of some disease states are mentioned.

BH3‐only proteins are part of a regulatory network that control the sustained signalling of the unfolded protein response sensor IRE1α

Adaptation to endoplasmic reticulum (ER) stress depends on the activation of the unfolded protein response (UPR) stress sensor inositol‐requiring enzyme 1α (IRE1α), which functions as an endoribonuclease that splices the mRNA of the transcription factor XBP‐1 (X‐box‐binding protein‐1). Through a global proteomic approach we identified the BCL‐2 family member PUMA as a novel IRE1α interactor. Immun oprecipitation experiments confirmed this interaction and further detected the association of IRE1α with BIM, another BH3‐only protein. BIM and PUMA double‐knockout cells failed to maintain sustained XBP‐1 mRNA splicing after prolonged ER stress, resulting in early inactivation. Mutation in the BH3 domain of BIM abrogated the physical interaction with IRE1α, inhibiting its effects on XBP‐1 mRNA splicing. Unexpectedly, this regulation required BCL‐2 and was antagonized by BAD or the BH3 domain mimetic ABT‐737. The modulation of IRE1α RNAse activity by BH3‐only proteins was recapitulated in a cell‐free system suggesting a direct regulation. Moreover, BH3‐only proteins controlled XBP‐1 mRNA splicing in vivo and affected the ER stress‐regulated secretion of antibodies by primary B cells. We conclude that a subset of BCL‐2 family members participates in a new UPR‐regulatory network, thus assuming apoptosis‐unrelated functions.

Prion protein misfolding affects calcium homeostasis and sensitizes cells to endoplasmic reticulum stress.

Prion-related disorders (PrDs) are fatal neurodegenerative disorders characterized by progressive neuronal impairment as well as the accumulation of an abnormally folded and protease resistant form of the cellular prion protein, termed PrPRES. Altered endoplasmic reticulum (ER) homeostasis is associated with the occurrence of neurodegeneration in sporadic, infectious and familial forms of PrDs. The ER operates as a major intracellular calcium store, playing a crucial role in pathological events related to neuronal dysfunction and death. Here we investigated the possible impact of PrP misfolding on ER calcium homeostasis in infectious and familial models of PrDs. Neuro2A cells chronically infected with scrapie prions showed decreased ER-calcium content that correlated with a stronger upregulation of UPR-inducible chaperones, and a higher sensitivity to ER stress-induced cell death. Overexpression of the calcium pump SERCA stimulated calcium release and increased the neurotoxicity observed after exposure of cells to brain-derived infectious PrPRES. Furthermore, expression of PrP mutants that cause hereditary Creutzfeldt-Jakob disease or fatal familial insomnia led to accumulation of PrPRES and their partial retention at the ER, associated with a drastic decrease of ER calcium content and higher susceptibility to ER stress. Finally, similar results were observed when a transmembrane form of PrP was expressed, which is proposed as a neurotoxic intermediate. Our results suggest that alterations in calcium homeostasis and increased susceptibility to ER stress are common pathological features of both infectious and familial PrD models.

Transient receptor potential melastatin 4 inhibition prevents lipopolysaccharide-induced endothelial cell death

AIMS Endothelial dysfunction is decisive in the progression of cardiovascular diseases. Lipopolysaccharide (LPS)-induced reactive oxygen species (ROS)-mediated endothelial cell death is a main feature observed in inflammation secondary to endotoxaemia, emerging as a leading cause of death among critically ill patients in intensive care units. However, the molecular mechanism underlying LPS-induced endothelial cell death is not well understood. Transient receptor protein melastatin 4 (TRPM4) is an ion channel associated with cell death that is expressed in endothelium and modulated by ROS. Here, we investigate the role of TRPM4 in LPS-induced endothelial cell death, testing whether suppression of the expression of TRPM4 confers endothelial cell resistance to LPS challenge. METHODS AND RESULTS Using primary cultures of human umbilical vein endothelial cells (HUVEC), we demonstrate that TRPM4 is critically involved in LPS-induced endothelial cell death. HUVEC exposed to LPS results in Na(+)-dependent cell death. Pharmacological inhibition of TRPM4 with 9-phenanthrol or glibenclamide protects endothelium against LPS exposure for 48 h. Furthermore, TRPM4-like currents increase in cells pre-treated with LPS and inhibited with glibenclamide. Of note, suppression of TRPM4 expression by siRNA or suppression of its activity in a dominant negative mutant is effective in decreasing LPS-induced endothelial cell death when cells are exposed to LPS for 24-30 h. CONCLUSION TRPM4 is critically involved in LPS-induced endothelial cell death. These results demonstrate that either pharmacological inhibition of TRPM4, suppression of TRPM4 expression, or inhibition of TRPM4 activity are able to protect endothelium against LPS injury. These results are useful in sepsis drug design and development of new strategies for sepsis therapy.

Endovenous laser and echo-guided foam ablation in great saphenous vein reflux: one-year follow-up results

BACKGROUND Great saphenous vein (GSV) reflux is the most frequent form of venous insufficiency in symptomatic patients and is commonly responsible for varicose veins of the lower extremity. This non-randomized prospective controlled study was designed to test the hypothesis that 1) endovenous laser treatment is more effective than foam sclerotherapy in the closure of the refluxing GSV (as measured by degree of great saphenous vein reflux and venous clinical severity score changes) and 2) to record the associated complications of echo-guided endovenous chemical ablation with foam and endovenous laser therapy for the treatment of great saphenous vein reflux and to further identify risk factors associated with treatment failure. METHODS Between January 1, 2006 and June 25, 2006, patients seeking treatment of varicose veins at a private practice of vascular medicine were assessed for the study. Inclusion criteria were: 1) presence of great saphenous vein reflux and 2) C2-6, Epr, A s, according to the CEAP classification. The selected patients consented into the study and were allowed to choose between foam (53 patients) or laser (45 patients) treatment. Duplex examinations were performed prior to treatment and at seven and 14 days, four weeks, six months, and one year after treatment. Venous clinical severity score was assessed pre-treatment and at one year post-procedure. RESULTS The cohorts showed no statistically significant differences in age, sex, clinical and anatomical presentation, great saphenous vein diameter, and venous clinical severity score before the treatments. After one year follow up, occlusion of the great saphenous vein was confirmed in 93.4% (42/45) of limbs studied in the laser group and 77.4% (41/53) of limbs in the foam group (P < .0465). Venous clinical severity score significantly improved in both groups (P < .0001). Procedure associated pain was higher in the laser group (P < .0082). Induration, phlebitis, and ecchymosis were the most common complications. Logistical regression and subgroups analysis shown that a larger great saphenous vein diameter measured before treatment was associated with treatment failure in the foam (odds ratio 1.68, 95% CI 1.24-2.27, P < .0008) and in the laser group (odds ratio 1.91, 95% CI 1.02-3.59, P < .0428). A 90% treatment success is predicted for veins <6.5 mm in the foam group versus veins <12 mm in the laser group. CONCLUSIONS Overall, endovenous laser ablation achieved higher occlusion rates than echo-guided chemical ablation with foam after one year follow-up. Matching the patient to the technique based on great saphenous vein diameter measured before treatment may assist in boosting the treatment success rate to >90%. A larger patient cohort followed and compared over a longer period of time would be required to confirm these findings.

TMBIM3/GRINA is a novel unfolded protein response (UPR) target gene that controls apoptosis through the modulation of ER calcium homeostasis.

Transmembrane BAX inhibitor motif-containing (TMBIM)-6, also known as BAX-inhibitor 1 (BI-1), is an anti-apoptotic protein that belongs to a putative family of highly conserved and poorly characterized genes. Here we report the function of TMBIM3/GRINA in the control of cell death by endoplasmic reticulum (ER) stress. Tmbim3 mRNA levels are strongly upregulated in cellular and animal models of ER stress, controlled by the PERK signaling branch of the unfolded protein response. TMBIM3/GRINA synergies with TMBIM6/BI-1 in the modulation of ER calcium homeostasis and apoptosis, associated with physical interactions with inositol trisphosphate receptors. Loss-of-function studies in D. melanogaster demonstrated that TMBIM3/GRINA and TMBIM6/BI-1 have synergistic activities against ER stress in vivo. Similarly, manipulation of TMBIM3/GRINA levels in zebrafish embryos revealed an essential role in the control of apoptosis during neuronal development and in experimental models of ER stress. These findings suggest the existence of a conserved group of functionally related cell death regulators across species beyond the BCL-2 family of proteins operating at the ER membrane.

Hydrogen Peroxide Removes TRPM4 Current Desensitization Conferring Increased Vulnerability to Necrotic Cell Death

Necrosis is associated with an increase in plasma membrane permeability, cell swelling, and loss of membrane integrity with subsequent release of cytoplasmic constituents. Severe redox imbalance by overproduction of reactive oxygen species is one of the main causes of necrosis. Here we demonstrate that H2O2 induces a sustained activity of TRPM4, a Ca2+-activated, Ca2+-impermeant nonselective cation channel resulting in an increased vulnerability to cell death. In HEK 293 cells overexpressing TRPM4, H2O2 was found to eliminate in a dose-dependent manner TRPM4 desensitization. Site-directed mutagenesis experiments revealed that the Cys1093 residue is crucial for the H2O2-mediated loss of desensitization. In HeLa cells, which endogenously express TRPM4, H2O2 elicited necrosis as well as apoptosis. H2O2-mediated necrosis but not apoptosis was abolished by replacement of external Na+ ions with sucrose or the non-permeant cation N-methyl-d-glucamine and by knocking down TRPM4 with a shRNA directed against TRPM4. Conversely, transient overexpression of TRPM4 in HeLa cells in which TRPM4 was previously silenced re-established vulnerability to H2O2-induced necrotic cell death. In addition, HeLa cells exposed to H2O2 displayed an irreversible loss of membrane potential, which was prevented by TRPM4 knockdown.

Phosphorylation of AKT/PKB by CK2 is necessary for the AKT-dependent up-regulation of β-catenin transcriptional activity

β‐Catenin is a key protein in the canonical Wnt signaling pathway and in many cancers alterations in transcriptional activity of its components are observed. This pathway is up‐regulated by the protein kinase CK2, but the underlying mechanism of this change is unknown. It has been demonstrated that CK2 hyperactivates AKT/PKB by phosphorylation at Ser129, and AKT phosphorylates β‐catenin at Ser552, which in turn, promotes its nuclear localization and transcriptional activity. However, the consequences of CK2‐dependent hyperactivation of AKT on β‐catenin activity and cell viability have not been evaluated. We assessed this regulatory process by manipulating the activity of CK2 and AKT through overexpression of wild‐type, constitutively active and dominant negative forms of these proteins as well as analyzing β‐catenin‐dependent transcriptional activity, survivin expression and viability in HEK‐293T cells. We observed that CK2α overexpression up‐regulated the β‐catenin transcriptional activity, which correlated to an increased nuclear localization of β‐catenin as well as survivin expression. Importantly, these effects were strongly reversed when an AKT‐S129A mutant was co‐expressed in the same cells, followed by a significant decrease in cell viability but no changes in β‐catenin stability. Taken together, the data suggest that the CK2α‐dependent up‐regulation of β‐catenin activity requires phosphorylation of AKT in human embryonic kidney cells. J. Cell. Physiol. 226: 1953–1959, 2011.

ALS-linked protein disulfide isomerase variants cause motor dysfunction

Disturbance of endoplasmic reticulum (ER) proteostasis is a common feature of amyotrophic lateral sclerosis (ALS). Protein disulfide isomerases (PDIs) are ER foldases identified as possible ALS biomarkers, as well as neuroprotective factors. However, no functional studies have addressed their impact on the disease process. Here, we functionally characterized four ALS‐linked mutations recently identified in two major PDI genes, PDIA1 and PDIA3/ERp57. Phenotypic screening in zebrafish revealed that the expression of these PDI variants induce motor defects associated with a disruption of motoneuron connectivity. Similarly, the expression of mutant PDIs impaired dendritic outgrowth in motoneuron cell culture models. Cellular and biochemical studies identified distinct molecular defects underlying the pathogenicity of these PDI mutants. Finally, targeting ERp57 in the nervous system led to severe motor dysfunction in mice associated with a loss of neuromuscular synapses. This study identifies ER proteostasis imbalance as a risk factor for ALS, driving initial stages of the disease.

TRPM4 enhances cell proliferation through up‐regulation of the β‐catenin signaling pathway

Altered expression of some members of the TRP ion channel superfamily has been associated with the development of pathologies like cancer. In particular, TRPM4 levels are reportedly elevated in diffuse large B‐cell non‐Hodgkin lymphoma, prostate, and cervical cancer. However, whether such changes in TRPM4 expression may be relevant to genesis or progression of cancer remains unknown. Here we show that reducing TRPM4 expression decreases proliferation of HeLa cells, a cervical cancer‐derived cell line. In this cell line, constitutive TRPM4 silencing promoted GSK‐3β‐dependent degradation of β‐catenin and reduced β‐catenin/Tcf/Lef‐dependent transcription. Conversely, overexpression of TRPM4 in T‐REx 293 cells (a HEK293‐derived cell line) increased cell proliferation and β‐catenin levels. Our results identify TRPM4 as an important, unanticipated regulator of the β‐catenin pathway, where aberrant signaling is frequently associated with cancer. J. Cell. Physiol. 226: 103–109, 2010.