The mechanism of activation and repression of apoptosis has been a central focus of many studies examining the role of programmed cell death in both normal and pathological conditions. Despite ...intensive research efforts, the precise cellular and molecular mechanisms that trigger and/or prevent apoptosis remain undefined. A universal characteristic of apoptosis is the loss of cell volume or cell shrinkage, recently termed apoptotic volume decrease. While cell shrinkage has traditionally been viewed as a passive event during apoptosis, recent work from several laboratories has shown that the loss of cell volume, or more specifically the flux of ions associated with the change in cell size, play a critical role in the regulation of the cell death machinery. On going studies continue to support the hypothesis that the change in intracellular ions can alter a cells decision to die by apoptosis.
The movement of intracellular monovalent cations has previously been shown to play a critical role in events leading to the characteristics associated with apoptosis. A loss of intracellular ...potassium and sodium occurs during apoptotic cell shrinkage establishing an intracellular environment favorable for nuclease activity and caspase activation. We have now investigated the potential movement of monovalent ions in Jurkat cells that occur prior to cell shrinkage following the induction of apoptosis. A rapid increase in intracellular sodium occurs early after apoptotic stimuli suggesting that the normal negative plasma membrane potential may change during cell death. We report here that diverse apoptotic stimuli caused a rapid cellular depolarization of Jurkat T-cells that occurs prior to and after cell shrinkage. In addition to the early increase in intracellular Na+, 86Rb+studies reveal a rapid inhibition of K+ uptake in response to anti-Fas. These effects on Na+ and K+ ions were accounted for by the inactivation of the Na+/K+-ATPase protein and its activity. Furthermore, ouabain, a cardiac glycoside inhibitor of the Na+/K+-ATPase, potentiated anti-Fas-induced apoptosis. Finally, activation of an anti-apoptotic signal,i.e. protein kinase C, prevented both cellular depolarization in response to anti-Fas and all downstream characteristics associated with apoptosis. Thus cellular depolarization is an important early event in anti-Fas-induced apoptosis, and the inability of cells to repolarize via inhibition of the Na+/K+-ATPase is a likely regulatory component of the death process.
Evidence shows that the CD38 molecule, recently involved in the two main features of asthma, bronchial hyper-responsiveness and airway inflammation, could represent a new potential therapeutic target ...for asthma. In this study, we investigated whether glucocorticoid (GC), the most effective treatment for lung diseases, can affect CD38 expression in human airway smooth muscle (ASM) cells treated with different pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNFalpha) and interferons (IFNs). We found that CD38 expression induced by TNFalpha alone was completely abrogated by fluticasone (100 nM), dexamethasone (1 microM), or budesonide (100 nM). In contrast, the synergistic induction of CD38 by the combination of TNFalpha with IFNgamma or IFNbeta, but not with IL-1beta or IL-13, was completely insensitive to the GC inhibitory effects. We also found that TNFalpha and IFNgamma impaired GC responsiveness by inhibiting steroid induced both 1) GRalpha-DNA binding activity and 2) GC-responsive element-(GRE)-dependent gene transcription. Although levels of the GC receptor (GR) alpha isoform remained unchanged, expression of GRbeta, the dominant-negative GR isoform, was synergistically increased by TNFalpha and IFNgamma with a GRalpha/GRbeta ratio of 1 to 3. More importantly, fluticasone failed to induce GRE-dependent gene transcription and to suppress TNFalpha-induced CD38 expression in ASM cells transfected with constitutively active GRbeta. We conclude that, upon pro-inflammatory cytokine stimulation, CD38 expression becomes insensitive to GC action by a mechanism involving the up-regulation of GRbeta isoform, thus providing a novel in vitro cellular model to dissect GC resistance in primary cells.
Apoptosis is a form of regulated cell death (RCD) that involves proteases of the caspase family. Pharmacological and genetic strategies that experimentally inhibit or delay apoptosis in mammalian ...systems have elucidated the key contribution of this process not only to (post-)embryonic development and adult tissue homeostasis, but also to the etiology of multiple human disorders. Consistent with this notion, while defects in the molecular machinery for apoptotic cell death impair organismal development and promote oncogenesis, the unwarranted activation of apoptosis promotes cell loss and tissue damage in the context of various neurological, cardiovascular, renal, hepatic, infectious, neoplastic and inflammatory conditions. Here, the Nomenclature Committee on Cell Death (NCCD) gathered to critically summarize an abundant pre-clinical literature mechanistically linking the core apoptotic apparatus to organismal homeostasis in the context of disease.
Alternative splicing of the human glucocorticoid receptor gene generates a nonhormone binding splice variant (hGRbeta) that differs from the wild-type receptor (hGRalpha) only at the carboxyl ...terminus. Previously we have shown that hGRbeta inhibits the transcriptional activity of hGRalpha, which is consistent with reports of elevated hGRbeta expression in patients with generalized and tissue-specific glucocorticoid resistance. The potential role of hGRbeta in the regulation of target cell sensitivity to glucocorticoids prompted us to further evaluate its dominant negative activity in other model systems and to investigate its mode of action. We demonstrate in multiple cell types that hGRbeta inhibits hGRalpha-mediated activation of the mouse mammary tumor virus promoter. In contrast, the ability of the progesterone and androgen receptors to activate this promoter is only weakly affected by hGRbeta. hGRbeta also inhibits hGRalpha-mediated repression of an NF-kappaB-responsive promoter but does not interfere with homologous down-regulation of hGRalpha. We show that hGRbeta can associate with the heat shock protein hsp90 although with lower affinity than hGRalpha. In addition, hGRbeta binds GRE-containing DNA with a greater capacity than hGRalpha in the absence of glucocorticoids. Glucocorticoid treatment enhances hGRalpha, but not hGRbeta, binding to DNA. Moreover, we demonstrate that hGRalpha and hGRbeta can physically associate with each other in a heterodimer. Finally, we show that the dominant negative activity of hGRbeta resides within its unique carboxyl-terminal 15 amino acids. Taken together, our results suggest that formation of transcriptionally impaired hGRalpha-hGRbeta heterodimers is an important component of the mechanism responsible for the dominant negative activity of hGRbeta.
Apoptosis is an active process with distinct features including loss of cell volume, chromatin condensation, internucleosomal DNA fragmentation, and apoptotic body formation. Among the classical ...characteristics that define apoptosis, the loss of cell volume has become a very important component of the programmed cell death process. Changes in cell volume result from alterations in the homeostasis of ions and in particular the movement of Na+ and K+ ions. Most living cells have a high concentration of intracellular K+ and a low concentration of intracellular Na+. This is in contrast to the outside of the cell, where there is a high concentration of extracellular Na+ and a low concentration of extracellular K+. Thus a concentration gradient exists for the loss and gain of intracellular K+ and Na+, respectively. This gradient is maintained through the activity of various ionic channels and transporters, but predominantly the activity of the Na+/K+‐ATPase. During apoptosis, there is compelling evidence indicating an early increase in intracellular Na+ followed by a decrease in both intracellular K+ and Na+ suggesting a regulatory role for these cations during both the initial signalling, and the execution phase of apoptosis. Recent studies have shown that the Na+/K+‐ATPase is involved in controlling perturbations of Na+ and K+ homeostasis during apoptosis, and that anti‐apoptotic Bcl‐2 and Bcl‐XL molecules influence these ionic fluxes. Finally, understanding the regulation or deregulation of ionic homeostasis during apoptosis is critical to facilitate the treatment of cardiovascular, neurological, and renal diseases where apoptosis is known to play a major role.
Alternative splicing of the human glucocorticoid receptor (hGR) primary transcript produces two receptor isoforms, hGRalpha and hGRbeta, which differ at their carboxyl termini. The hGRalpha isoform ...conveys endocrine information to target tissues by altering patterns of gene expression in a hormone-dependent fashion. In contrast to hGRalpha, very little is known about the hGRbeta splice variant. Using hGRalpha- and hGRbeta-specific riboprobes on human multiple tissue Northern blots, we show that the hGRbeta message has a widespread tissue distribution. We also prove by reverse transcriptase-polymerase chain reaction that the alternative splicing event underlying the formation of the hGRbeta message occurs in these tissues. Because the hGRbeta protein differs from hGRalpha at the extreme COOH terminus, we investigated several of the biochemical properties of hGRbeta expressed in transfected cells. hGRbeta does not bind the glucocorticoid agonist dexamethasone nor the glucocorticoid antagonist RU38486 in vivo. Moreover, in contrast to hGRalpha, hGRbeta is located primarily in the nucleus of transfected cells independent of hormone administration. Finally, in the absence of hGRalpha, hGRbeta is transcriptionally inactive on a glucocorticoid-responsive enhancer. However, when both isoforms are expressed in the same cell, hGRbeta inhibits the hormone-induced, hGRalpha-mediated stimulation of gene expression. Thus, hGRbeta potentially functions as a dominant negative inhibitor of hGRalpha activity.
It is hoped that this review will give the reader a taste of some of the mechanisms used by the glucocorticoid receptor to repress gene function. These mechanisms include direct binding to DNA, ...antagonism of other transcription factor families and sequestration of necessary cofactors. Each of these mechanisms, and others, are discussed.
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