Susana Castro-Obregon

Function of reactive oxygen species during animal development : Passive or active?

Oxidative stress is considered causal of aging and pathological cell death, however, very little is known about its function in the natural processes that support the formation of an organism. It is generally thought that cells must continuously protect themselves from the possible damage caused by reactive oxygen species (ROS) (passive ROS function). However, presently, ROS are recognized as physiologically relevant molecules that mediate cell responses to a variety of stimuli, and the activities of several molecules, some developmentally relevant, are directly or indirectly regulated by oxidative stress (active ROS function). Here we review recent data that are suggestive of specific ROS functions during development of animals, particularly mammals.

Molecular components of a cell death pathway activated by endoplasmic reticulum stress

Alterations in Ca2+ homeostasis and accumulation of misfolded proteins in the endoplasmic reticulum (ER) cause ER stress that ultimately leads to programmed cell death. Recent studies have shown that ER stress triggers programmed cell death via an alternative intrinsic pathway of apoptosis that, unlike the intrinsic pathway described previously, is independent of Apaf-1 and cytochrome c. In the present work, we have used a set of complementary approaches, including two-dimensional gel electrophoresis coupled with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry and nano-liquid chromatography-electrospray ionization mass spectrometry with tandem mass spectrometry, RNA interference, co-immunoprecipitation, immunodepletion of candidate proteins, and reconstitution studies, to identify mediators of the ER stress-induced cell death pathway. Our data identify two molecules, valosin-containing protein and apoptosis-linked gene-2 (ALG-2), that appear to play a role in mediating ER stress-induced cell death.

Reactive oxygen species: A radical role in development?

Reactive oxygen species (ROS), mostly derived from mitochondrial activity, can damage various macromolecules and consequently cause cell death. This ROS activity has been characterized in vitro, and correlative evidence suggests a role in various pathological conditions. In addition to this passive ROS activity, ROS also participate in cell signaling processes, though the relevance of this function in vivo is poorly understood. Throughout development, elevated cell activity is probably accompanied by highly active metabolism and, consequently, the production of large amounts of ROS. To allow proper development, cells must protect themselves from these potentially damaging ROS. However, to what degree ROS could participate as signaling molecules controlling fundamental and developmentally relevant cellular processes such as proliferation, differentiation, and death is an open question. Here we discuss why available data do not yet provide conclusive evidence on the role of ROS in development, and we review recent methods to detect ROS in vivo and genetic strategies that can be exploited specifically to resolve these uncertainties.

A ligand-receptor pair that triggers a non-apoptotic form of programmed cell death

Several receptors that mediate apoptosis have been identified, such as Fas and tumor necrosis factor receptor I. Studies of the signal transduction pathways utilized by these receptors have played an important role in the understanding of apoptosis. Here we report the first ligand-receptor pair–the neuropeptide substance P and its receptor, neurokinin-1 receptor (NK1R)–that mediates an alternative, non-apoptotic form of programmed cell death. This pair is widely distributed in the central and peripheral nervous systems, and has been implicated in pain mediation and depression, among other effects. Here we demonstrate that substance P induces a non-apoptotic form of programmed cell death in hippocampal, striatal, and cortical neurons. This cell death requires gene expression, displays a non-apoptotic morphology, and is independent of caspase activation. The same form of cell death is induced by substance P in NK1R-transfected human embryonic kidney cells. These results argue that NK1R activates a death pathway different than apoptosis, and provide a signal transduction system by which to study an alternative, non-apoptotic cell death program.

Role of retinoic acid and oxidative stress in embryonic stem cell death and neuronal differentiation

Embryonic stem (ES) cells are a suitable system to study events occurring during development. In the present work we show that the apoptotic program was activated in ES cells, either by simple removal of the reducing agent 2‐mercapthoethanol (2‐ME), or by addition of all trans‐retinoic acid (ATRA) to embryoid bodies. In these two conditions, there was an increase in reactive oxygen species and antioxidants such as catalase, superoxide dismutase or phenol prevented ATRA‐induced cell death. Neuronal differentiation was observed when undifferentiated ES cells were treated with ATRA in the absence of serum and the presence of 2‐ME.

Interfering with multimerization of netrin-1 receptors triggers tumor cell death

Netrin-1 was recently proposed to control tumorigenesis by inhibiting apoptosis induced by the dependence receptors DCC (Deleted in colorectal cancer) and UNC5H. Although the loss of these dependence receptors’ expression has been described as a selective advantage for tumor growth and progression in numerous cancers, recent observations have shown that some tumors may use an alternative strategy to block dependence receptor-induced programmed cell death: the autocrine expression of netrin-1. This alternative strategy has been observed in a large fraction of aggressive breast cancers, neuroblastoma, pancreatic adenocarcinoma, and lung cancer. This observation is of potential interest regarding future targeted therapy, as in such cases interfering with the ability of netrin-1 to inhibit DCC or UNC5H-induced cell death is associated with apoptosis of netrin-1-expressing tumor cells in vitro, and with inhibition of tumor growth or metastasis in different animal tumor models. The understanding of the mechanism by which netrin-1 inhibits cell death is therefore of interest. Here, we show that netrin-1 triggers the multimerization of both DCC and UNC5H2 receptors, and that multimerization of the intracellular domain of DCC and UNC5H2 is the critical step to inhibit the proapoptotic effects of both of these receptors. Taking advantage of this property, we utilized a recombinant specific domain of DCC that (i) interacts with netrin-1 and (ii) inhibits netrin-1-induced multimerization, to trigger apoptosis in netrin-dependent tumor cells.

Downregulation of VRK1 by p53 in Response to DNA Damage Is Mediated by the Autophagic Pathway

Human VRK1 induces a stabilization and accumulation of p53 by specific phosphorylation in Thr18. This p53 accumulation is reversed by its downregulation mediated by Hdm2, requiring a dephosphorylated p53 and therefore also needs the removal of VRK1 as stabilizer. This process requires export of VRK1 to the cytosol and is inhibited by leptomycin B. We have identified that downregulation of VRK1 protein levels requires DRAM expression, a p53-induced gene. DRAM is located in the endosomal-lysosomal compartment. Induction of DNA damage by UV, IR, etoposide and doxorubicin stabilizes p53 and induces DRAM expression, followed by VRK1 downregulation and a reduction in p53 Thr18 phosphorylation. DRAM expression is induced by wild-type p53, but not by common human p53 mutants, R175H, R248W and R273H. Overexpression of DRAM induces VRK1 downregulation and the opposite effect was observed by its knockdown. LC3 and p62 were also downregulated, like VRK1, in response to UV-induced DNA damage. The implication of the autophagic pathway was confirmed by its requirement for Beclin1. We propose a model with a double regulatory loop in response to DNA damage, the accumulated p53 is removed by induction of Hdm2 and degradation in the proteasome, and the p53-stabilizer VRK1 is eliminated by the induction of DRAM that leads to its lysosomal degradation in the autophagic pathway, and thus permitting p53 degradation by Hdm2. This VRK1 downregulation is necessary to modulate the block in cell cycle progression induced by p53 as part of its DNA damage response.

Progressive interdigital cell death: regulation by the antagonistic interaction between fibroblast growth factor 8 and retinoic acid

The complete cohort of molecules involved in interdigital cell death (ICD) and their interactions are yet to be defined. Bmp proteins, retinoic acid (RA) and Fgf8 have been previously identified as relevant factors in the control of ICD. Here we determined that downregulation of Fgf8 expression in the ectoderm overlying the interdigital areas is the event that triggers ICD, whereas RA is the persistent cell death-inducing molecule that acts on the distal mesenchyme by a mechanism involving the induction of Bax expression. Inhibition of the mitogen-activated protein kinase (Mapk) pathway prevents the survival effect of Fgf8 on interdigital cells and the accompanying Erk1/2 phosphorylation and induction of Mkp3 expression. Fgf8 regulates the levels of RA by both decreasing the expression of Raldh2 and increasing the expression of Cyp26b1, whereas RA reduces Fgfr1 expression and Erk1/2 phosphorylation. In the mouse limb, inhibition of Bmp signaling in the mesenchyme does not affect ICD. However, noggin in the distal ectoderm induces Fgf8 expression and reduces interdigit regression. In the chick limb, exogenous noggin reduces ICD, but, when applied to the distal mesenchyme, this reduction is associated with an increase in Fgf8 expression. In agreement with the critical decline in Fgf8 expression for the activation of ICD, distal interdigital cells acquire a proximal position as interdigit regression occurs. We identified proliferating distal mesenchymal cells as those that give rise to the interdigital cells fated to die. Thus, ICD is determined by the antagonistic regulation of cell death by Fgf8 and RA and occurs through a progressive, rather than massive, cell death mechanism.

PLAIDD, a type II death domain protein that interacts with p75 neurotrophin receptor

We describe the cloning and characterization of a rat single transmembrane protein that is homologous to the common neurotrophin receptor p75NTR in its death domain and the transmembrane region but dissimilar outside these regions. We have dubbed this protein PLAIDD, for p75-like apoptosis-inducing death domain protein. PLAIDD messenger RNA, which is ubiquitously distributed, is highly expressed in the embryo, but downregulated in adult tissues. Alternative splicing within the extracellular region of PLAIDD generates four RNA species, but only two of them are translated, PLAIDD_L and PLAIDD_S (long and short isoforms, respectively). While the amino acid sequence of the intracellular region of PLAIDD displays 41% identity with the intracellular region of p75NTR, the extracellular region of PLAIDD does not reveal any homology with p75NTR. Overexpression of each isoform of PLAIDD led to cytotoxicity in superior cervical ganglion neurons and in human embryonic kidney 293T cells. Both isoforms of PLAIDD could be co-immunoprecipitated with p75NTR, suggesting an interaction between these molecules.

APAP, a sequence-pattern recognition approach identifies substance P as a potential apoptotic peptide

We have previously described a novel cancer chemotherapeutic approach based on the induction of apoptosis in targeted cells by homing pro‐apoptotic peptides. In order to improve this approach we developed a computational method (approach for detecting potential apoptotic peptides, APAP) to detect short PAPs, based on the prediction of the helical content of peptides, the hydrophobic moment, and the isoelectric point. PAPs are toxic against bacteria and mitochondria, but not against mammalian cells when applied extracellularly. Among other peptides, substance P was identified as a PAP and subsequently demonstrated to be a pro‐apoptotic peptide experimentally. APAP thus provides a method to detect and ultimately improve pro‐apoptotic peptides for chemotherapy.