Biomedical subjects

David S Ucker

Publications and source records attributed to David S Ucker.

11 recordsLinked to original sources

PubMed2006

Unique membrane interaction mode of group IIF phospholipase A2.

The mechanisms by which secretory phospholipases A(2) (PLA(2)s) exert cellular effects are not fully understood. Group IIF PLA(2) (gIIFPLA(2)) is a structurally unique secretory PLA(2) with a long C-terminal extension. Homology modeling suggests that the membrane-binding surface of this acidic PLA(2) contains hydrophobic residues clustered near the C-terminal extension. Vesicle leakage and monolayer penetration measurements showed that gIIFPLA(2) had a unique ability to penetrate and disrupt compactly packed monolayers and bilayers whose lipid composition recapitulates that of the outer plasma membrane of mammalian cells. Fluorescence imaging showed that gIIFPLA(2) could also readily enter and deform plasma membrane-mimicking giant unilamellar vesicles. Mutation analysis indicates that hydrophobic residues (Tyr(115), Phe(116), Val(118), and Tyr(119)) near the C-terminal extension are responsible for these activities. When gIIFPLA(2) was exogenously added to HEK293 cells, it initially bound to the plasma membrane and then rapidly entered the cells in an endocytosis-independent manner, but the cell entry did not lead to a significant degree of phospholipid hydrolysis. GIIFPLA(2) mRNA was detected endogenously in human CD4(+) helper T cells after in vitro stimulation and exogenously added gIIFPLA(2) inhibited the proliferation of a T cell line, which was not seen with group IIA PLA(2). Collectively, these data suggest that unique membrane-binding properties of gIIFPLA(2) may confer special functionality on this secretory PLA(2) under certain physiological conditions.

Amino Acid Sequence↗

PubMed2006

Specific recognition of apoptotic cells reveals a ubiquitous and unconventional innate immunity.

The purpose of physiological cell death is the noninflammatory clearance of cells that have become inappropriate or nonfunctional. Consistent with this function, the recognition of apoptotic cells by professional phagocytes, including macrophages and dendritic cells, triggers a set of potent anti-inflammatory responses manifest on multiple levels. The immediate-early inhibition of proinflammatory cytokine gene transcription in the phagocyte is a proximate consequence of recognition of the apoptotic corpse, independent of subsequent engulfment and soluble factor involvement. Here, we show that recognition is linked to a characteristic signature of responses, including MAPK signaling events and the ablation of proinflammatory transcription and cytokine secretion. Specific recognition and response occurs without regard to the origin (species, tissue type, or suicidal stimulus) of the apoptotic cell and does not involve Toll-like receptor signaling. These features mark this as an innate immunity fundamentally distinct from the discrimination of "self" versus "other" considered to be the hallmark of conventional immunity. This profound unconventional innate immune discrimination of effete from live cells is as ubiquitous as apoptotic cell death itself, manifest by professional and nonprofessional phagocytes and nonphagocytic cell types alike. Innate apoptotic immunity provides an intrinsic anti-inflammatory circuit that attenuates proinflammatory responses dynamically and may act systemically as a powerful physiological regulator of immunity.

Animals↗

PubMed2006

A mutant allele of BARA/LIN-9 rescues the cdk4-/- phenotype by releasing the repression on E2F-regulated genes.

It has been proposed that C. elegans LIN-9 functions downstream of CDK4 in a pathway that regulates cell proliferation. Here, we report that mammalian BARA/LIN-9 is a predominantly nuclear protein that inhibits cell proliferation. More importantly, we demonstrate that BARA/LIN-9 also acts downstream of cyclin D/CDK4 in mammalian cells since (i) its antiproliferative effect is partially blocked by coexpression of cyclin D1, and (ii) a mutant form that lacks the first 84 amino acids rescues several phenotypic alterations observed in mice null for cdk4. Interestingly, mutation of BARA/LIN-9 restores the expression of E2F target genes in CDK4 null MEFs, indicating that the wild-type protein plays a role in the expression of genes required for the G1/S transition.

Alleles↗

PubMed2005

Apoptotic cells, at all stages of the death process, trigger characteristic signaling events that are divergent from and dominant over those triggered by necrotic cells: Implications for the delayed clearance model of autoimmunity.

Current models of autoimmunity suggest that delayed clearance of apoptotic cells leads to the presentation of apoptotic antigens in the context of inflammatory signals, with resultant autoimmunity. These models implicitly assume that, in contrast to early apoptotic cells (that retain membrane integrity), late apoptotic cells (with compromised membranes) act like necrotic cells (which also lack intact membranes), possibly because of the release of proinflammatory intracellular contents. We showed previously that early apoptotic and necrotic cells induce distinct mitogen-activated protein kinase modules in macrophages with which they interact. Exposure to apoptotic cells led to nearly complete inhibition of both basal and macrophage colony-stimulating factor-induced ERK1/2 by macrophages. In contrast, necrotic cells induced ERK1/2. We show here that apoptotic cells also strongly induced both c-Jun N-terminal kinase and p38, whereas necrotic cells had no detectable effect on c-Jun N-terminal kinase and p38. We also compared the signaling events induced in macrophages by exposure to early apoptotic cells, late apoptotic cells, and necrotic cells. The signaling events induced by late apoptotic cells were identical to and just as potent as those induced by early apoptotic cells. Thus, apoptotic cells are functionally equivalent throughout the cell death process, irrespective of membrane integrity. Moreover, the effects of both early and late apoptotic cells on signaling were dominant over those of necrotic cells. These data show that apoptotic cells do not become proinflammatory upon the loss of membrane integrity and are inconsistent with the notion that delayed clearance alone can lead to autoimmunity.

Animals↗

PubMed2005

The presumptive phosphatidylserine receptor is dispensable for innate anti-inflammatory recognition and clearance of apoptotic cells.

The role of the presumptive phosphatidylserine receptor (PSR) in the recognition and engulfment of apoptotic cells, and the antiinflammatory response they exert, has been of great interest. Genetic deficiency of PSR in the mouse is lethal perinatally, and results to date have been ambiguous with regard to the phagocytic and inflammatory phenotypes associated with that deficiency. Recently, we found that the specific functional recognition of apoptotic cells is a ubiquitous property of virtually all cell types, including mouse embryo fibroblasts, and reflects an innate immunity that discriminates live from effete cells. Taking advantage of this property of fibroblasts, we generated, PSR(+/+), PSR(+/-), and PSR(-/-) fibroblast cell lines to examine definitively the involvement of PSR in apoptotic recognition and inflammatory modulation. Our data demonstrate that PSR-deficient cells are fully competent to recognize, engulf, and respond to apoptotic cells. Signal transduction in the responder cells, including the activation of Akt and Rac1, is unimpaired in the absence of PSR. We confirm as well that PSR is localized predominantly to the nucleus. However, it does not play a role in pro-inflammatory transcription or in the anti-inflammatory modulation of that transcriptional response triggered by apoptotic cells. We conclude that PSR is not involved generally in either specific innate recognition or engulfment of apoptotic cells.

Animals↗

PubMed2005

Inhibiting myosin light chain kinase induces apoptosis in vitro and in vivo.

Previous short-term studies have correlated an increase in the phosphorylation of the 20-kDa light chain of myosin II (MLC20) with blebbing in apoptotic cells. We have found that this increase in MLC20 phosphorylation is rapidly followed by MLC20 dephosphorylation when cells are stimulated with various apoptotic agents. MLC20 dephosphorylation is not a consequence of apoptosis because MLC20 dephosphorylation precedes caspase activation when cells are stimulated with a proapoptotic agent or when myosin light chain kinase (MLCK) is inhibited pharmacologically or by microinjecting an inhibitory antibody to MLCK. Moreover, blocking caspase activation increased cell survival when MLCK is inhibited or when cells are treated with tumor necrosis factor alpha. Depolymerizing actin filaments or detaching cells, processes that destabilize the cytoskeleton, or inhibiting myosin ATPase activity also resulted in MLC20 dephosphorylation and cell death. In vivo experiments showed that inhibiting MLCK increased the number of apoptotic cells and retarded the growth of mammary cancer cells in mice. Thus, MLC20 dephosphorylation occurs during physiological cell death and prolonged MLC20 dephosphorylation can trigger apoptosis.

Animals↗

PubMed2004

Innate immune discrimination of apoptotic cells: repression of proinflammatory macrophage transcription is coupled directly to specific recognition.

Physiological cell death is a process the purpose of which is the elimination of functionally inappropriate cells in a manner that does not elicit an inflammatory response. We have shown previously that the ability of apoptotic corpses to be recognized by macrophages and to modulate the proinflammatory responses of those cells represents paradoxically a gain-of-function acquired during the physiological cell death process. Cells that die pathologically (that is, necrotic vs apoptotic corpses) also are recognized by macrophages but do not down-regulate macrophage inflammatory responses; the recognition of these two classes of native dying cells occurs via distinct and noncompeting mechanisms. We have examined the apoptotic modulation of proinflammatory cytokine gene transcription in macrophages (by real-time RT-PCR analysis) and the corresponding modulation of transcriptional activators (by transcriptional reporter analyses). Our data demonstrate that apoptotic cells target the proinflammatory transcriptional machinery of macrophages with which they interact, without apparent effect on proximal steps of Toll-like receptor signaling. The modulatory activity of the corpse is manifest as an immediate-early inhibition of proinflammatory cytokine gene transcription, and is exerted directly upon binding to the macrophage, independent of subsequent engulfment and soluble factor involvement. Recognition and inflammatory modulation represent key elements of an innate immune response that discriminates live from effete cells, and without regard to self.

Animals↗

PubMed2003

Caspase activity is not sufficient to execute cell death.

Molecular studies of the physiological cell death process have focused attention on the role of effector caspases as critical common elements of the lethal mechanism. Diverse death signals act afferently via distinct signaling pathways to activate these resident proenzyme molecules post-translationally. Whether this molecular convergence represents the mechanistic point of irreversible commitment to cell death has not been established. That a number of caspase substrates are proteins that serve important roles in cellular homeostasis has led to the view that the acquisition of this activity must be the determinative step in cell death. Observations that caspases serve in a regulatory role to catalyze the appearance of new activities involved in orderly cellular dissolution challenge this model of death as a simple process of proteolytic destruction. We found previously that caspase-dependent nuclear cyclin dependent kinase 2 (Cdk2) activity appears to be necessary for cell death. Employing direct cytofluorimetric analyses of intracellular caspase activity and colony forming assays, we now show that transient blockade of caspase-dependent Cdk2 activity confers long-lived sparing from death on cells otherwise triggered to die and fully replete with caspase activity. These data demonstrate that caspases, while necessary for apoptosis, are not sufficient to exert lethality. Caspase activation per se does not represent an irreversible point of commitment to physiological cell death.

CDC2-CDC28 Kinases↗

PubMed2003

Schizosaccharomyces pombe Ddb1 is functionally linked to the replication checkpoint pathway.

Schizosaccharomyces pombe Ddb1 is homologous to the mammalian DDB1 protein, which has been implicated in damaged-DNA recognition and global genomic repair. However, a recent study suggested that the S. pombe Ddb1 is involved in cell division and chromosomal segregation. Here, we provide evidence that the S. pombe Ddb1 is functionally linked to the replication checkpoint control gene cds1. We show that the S. pombe strain lacking ddb1 has slow growth due to delayed replication progression. Flow cytometric analysis shows an extensive heterogeneity in DNA content. Furthermore, the Deltaddb1 strain is hypersensitive to UV irradiation in S phase and is unable to tolerate a prolonged replication block imposed by hydroxyurea. Interestingly, the Deltaddb1 strain exhibits a high level of the Cds1 kinase activity during passage through S phase. Moreover, mutation of the cds1 gene relieves the defects observed in Deltaddb1 strain. The results suggest that many of the defects observed in Deltaddb1 cells are linked to an aberrant activation of Cds1, and that Ddb1 is functionally linked to Cds1.

Checkpoint Kinase 2↗

PubMed2002

The effector phase of physiological cell death relies exclusively on the posttranslational activation of resident components.

Inhibitors of transcription and translation can protect cells from physiological cell deaths induced by a variety of stimuli. These observations have been taken to suggest that de novo macromolecular synthesis may be an essential component of the cell death process. Paradoxically, the same inhibitors, at higher concentrations, themselves trigger the death of cells. Previously, we have mapped a conserved and ordered sequence of events that exerts physiological cell death. Diverse signals converge to activate this lethal pathway, composed of a proteolytic cascade of caspases and subsequent cyclin-dependent kinases. Here we report that inhibitors of nuclear gene expression, when they block cell death, act upstream of this lethal process to prevent its activation. In contrast, when cell death is triggered by high doses of the inhibitors, these same essential molecules are activated, despite the essentially complete blockade of macromolecular synthesis. This inhibitor-induced death response is associated with the release of cytochrome c from mitochondria and the activation of apical caspase 9 and is blocked by overexpression of Bcl-2. These data demonstrate that all essential molecules that exert lethality already are resident within cells and are activated posttranslationally upon stimulation. De novo macromolecular synthesis pertains idiosyncratically only to upstream, modulatory elements of particular death responses.

Apoptosis↗

PubMed2002

Vitamin E succinate is a potent novel antineoplastic agent with high selectivity and cooperativity with tumor necrosis factor-related apoptosis-inducing ligand (Apo2 ligand) in vivo.

Alpha-tocopheryl succinate (alpha-TOS), a redox-inactive analogue of vitamin E, is a strong inducer of apoptosis, whereas alpha-tocopherol (alpha-TOH) lacks apoptogenic activity (J. Neuzil et al., FASEB J., 15: 403-415, 2001). Here we investigated the possible antineoplastic activities of alpha-TOH and alpha-TOS and further explored the potential of alpha-TOS as an antitumor agent. Using nude mice with colon cancer xenografts, we found that alpha-TOH exerted modest antitumor activity and acted by inhibiting tumor cell proliferation. In contrast, alpha-TOS showed a more profound antitumor effect, at both the level of inhibition of proliferation and induction of tumor cell apoptosis. alpha-TOS was nontoxic to normal cells and tissues, triggered apoptosis in p53(-/-) and p21(Waf1/Cip1(-/-)) cancer cells, and exerted a cooperative proapoptotic activity with tumor necrosis factor-related apoptosis-inducing ligand (Apo2 ligand) due to differences in proapoptotic signaling. Finally, alpha-TOS cooperated with tumor necrosis factor-related apoptosis-inducing ligand in suppression of tumor growth in vivo. Vitamin E succinate is thus a potent and highly specific anticancer agent and/or adjuvant of considerable therapeutic potential.

Animals↗